December 2022
Volume 63, Issue 13
Open Access
Cornea  |   December 2022
Insufficient Dose of ERCC8 Protein Caused by a Frameshift Mutation Is Associated With Keratoconus With Congenital Cataracts
Author Affiliations & Notes
  • Xiao-Dan Hao
    Institute for Translational Medicine, The Affiliated Hospital of Qingdao University, College of Medicine, Qingdao University, Qingdao, China
  • Yi-Zhi Yao
    Institute for Translational Medicine, The Affiliated Hospital of Qingdao University, College of Medicine, Qingdao University, Qingdao, China
  • Kai-Ge Xu
    Institute for Translational Medicine, The Affiliated Hospital of Qingdao University, College of Medicine, Qingdao University, Qingdao, China
  • Bin Dong
    Institute for Translational Medicine, The Affiliated Hospital of Qingdao University, College of Medicine, Qingdao University, Qingdao, China
  • Wen-Hua Xu
    Department of Inspection, Medical Faculty of Qingdao University, Qingdao, China
  • Jing-Jing Zhang
    Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University (Shandong Eye Hospital), Jinan, China
    State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao, China
  • Correspondence: Xiao-Dan Hao, Institute for Translational Medicine, The Affiliated Hospital of Qingdao University, College of Medicine, Qingdao University, No. 308, Ningxia Road, Qingdao 266021, China; [email protected]
  • Jing-Jing Zhang, Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University (Shandong Eye Hospital), Jinan 250021, China; [email protected]
Investigative Ophthalmology & Visual Science December 2022, Vol.63, 1. doi:https://doi.org/10.1167/iovs.63.13.1
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      Xiao-Dan Hao, Yi-Zhi Yao, Kai-Ge Xu, Bin Dong, Wen-Hua Xu, Jing-Jing Zhang; Insufficient Dose of ERCC8 Protein Caused by a Frameshift Mutation Is Associated With Keratoconus With Congenital Cataracts. Invest. Ophthalmol. Vis. Sci. 2022;63(13):1. https://doi.org/10.1167/iovs.63.13.1.

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Abstract

Purpose: The purpose of this study was to identify a new candidate gene for keratoconus and congenital cataracts and further investigate its underlying pathogenic mechanisms.

Methods: This study, using a Chinese family with keratoconus and congenital cataracts, 262 patients with sporadic keratoconus, and 20 patients with sporadic congenital cataract as subjects, used clinical and genetic analysis and in vitro cell experiments to detect genetic mutations and further investigate the underlying pathogenic mechanisms.

Results: We found that a novel frameshift mutation of ERCC8 (NM_000082.3: c.394-398del, p. L132Nfs*6) is responsible for familial keratoconus with congenital cataracts. This mutation showed co-segregation with the phenotype in the family. This was revealed in another patient with sporadic keratoconus, absent in the 210 unrelated health controls, and considered to be “disease-causing.” ERCC8 was expressed both in the cornea and lens. Through an in vitro cell experiment, we further demonstrated that the mutant proteins of ERCC8 were degraded and could lead to an insufficient dose of the ERCC8 protein. An insufficient dose reduced the DNA damage repair ability of human corneal fibroblast (HTK) and lens epithelial cells (HLEC) treated with hydrogen peroxide, leading to both cells showing higher DNA damage levels. In addition, it decreased cell viability, resulting in decreased collagen expression in HTK and increased apoptosis in HLEC via aberrant activation of the unfolded protein response. All these results suggested that ERCC8 plays an important role in the normal function of corneal stromal and lens epithelial cells.

Conclusions: Our study showed that ERCC8 is a new gene associated with keratoconus and congenital cataracts.

Chinese Abstract

Keratoconus (KC) is a progressive corneal ectatic disorder characterized by corneal ectasia and thinning, resulting in reduced vision, irregular astigmatism, and corneal scarring, eventually necessitating corneal transplantation.1 The worldwide prevalence of KC is approximately 1:2000.2 The onset of KC usually occurs during adolescence, then progresses with age and has a lifelong effect on patients.3 KC has a clear genetic tendency, and genetic factors play a critical role in its pathogenesis.4,5 Recently, the use of high-throughput omics techniques has promoted the advancement of KC genetics. Until now, more than 80 KC-associated genes or regions have been identified by linkage analysis, genomewide association studies (GWAS), whole exome or genome sequencing (WES or WGS), or candidate gene association studies.6 However, owing to the genetic heterogeneity among patients with KC in different populations, the genetic etiology of most cases has not been effectively identified, and new candidate genes need to be explored. 
High-quality families represent extremely good material for genetic disease research, can often bring very valuable insights to genetic etiology research, and can lead to a breakthrough in the identification of pathogenic genes in many human genetic diseases.79 A high-quality KC pedigree is very rare, and most patients with KC are sporadic.2,4,5 Nevertheless, some inherited diseases are complicated by KC symptoms, such as brittle cornea syndrome,10 Wolfram syndrome,11 Leber's congenital amaurosis,12 and early-onset anterior polar cataracts,13 among others. Some well-known KC candidate genes, such as ZNF469, miR-184, TGFBI, and ZEB1, have been identified in families with these related diseases in the past, bringing valuable insights into KC genetic etiology.6,1318 
This study collected a pedigree with reports of KC made complicated by congenital cataracts, which provided valuable samples for the study of the genetic mechanism of KC. The WES analysis of patients is an effective strategy for identifying candidate pathogenic gene mutations. The screening of candidate gene mutations in more than 200 patients with KC and healthy controls was conducted to further determine the pathogenicity of candidate gene mutations. In vitro cell experiments were performed to study the functional changes and pathogenic mechanisms of candidate gene mutations. Finally, the findings of this study identified a new disease gene (ERCC8) underlying KC and congenital cataracts and provided insight into the insufficient dose of the ERCC8 protein in human corneal degeneration and lens cell apoptosis. These results will contribute to making accurate diagnoses and will provide new targets for the treatment of KC and congenital cataracts. 
Methods
Subject Recruitment and Clinical Examination
This study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Shandong Eye Hospital (Jinan, China). A Chinese family with KC with congenital cataracts, 262 additional patients with sporadic KC, 20 patients with sporadic congenital cataract, and 210 unrelated healthy controls were recruited from this hospital. Written informed consent was obtained from all participants (or guardians). A Whole Blood Genomic DNA Purification Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract genomic DNA. 
WES and Data Analysis
We conducted WES in the proband (I-2) of the family and her son (II-1) to identify the causal gene. Agilent SureSelect Human All Exon V6 (Agilent, Santa Clara, CA, USA) was used for exome capture. The IlluminaHiseq 2500 platform (Illumina Inc., San Diego, CA, USA) was used for sequencing. We then conducted data analysis and filtering according to the filtering strategy described in a previous study.19 The variants were classified according to the American College of Medical Genetics and Genomics (ACMG) variant-classification guidelines. Finally, the splice, non-synonymy, termination, and frameshift variants occurring in exons or located in canonical splicing sites, with a minor allele frequency <1% in database-single-nucleotide polymorphism (dbSNP), HapMap, and the 1000 Genomes Project databases and damaging functional prediction, which were classified as pathogenic or likely pathogenic and co-segregated with the phenotype in this family, were considered to be candidate causal variations. The variants in the genes reported to be associated with KC or cataracts have the priority of verification. 
ERCC8 Sequencing, Genotyping, and Protein Structure Prediction
The novel frameshift mutation of ERCC8 was genotyped in 262 additional patients with sporadic KC and 210 unrelated healthy controls using high-resolution melt (HRM) analysis, as described in a previous study.19 Sanger sequencing of ERCC8 was performed to confirm the results obtained using WES and HRM. Sanger sequencing of ERCC8 was also performed in 20 unrelated patients with congenital cataract for further cataract-related ERCC8 variant detection. The primers used for sequencing and genotyping the mutation of ERCC8 are shown in Supplementary Table S1
Furthermore, MEGA software was used to perform multiple protein sequence alignments among various species.20 MutationTaster (http://www.mutationtaster.org/) was applied to test the possible effect of amino acid substitution on protein function.21 The tertiary structure of mutant proteins was predicted using HOPE online software (http://www.cmbi.umcn.nl/hope).22 
ERCC8 Expression Assays in Mouse Eyes
ERCC8 protein immunofluorescence staining was performed on mouse eyeballs, as described in prior research,23 and then examined under a Nikon DS-Ri2 microscope (Nikon, Tokyo, Japan). Total RNA was extracted from the heart, liver, lungs, cornea, lens, conjunctiva, and retina tissues of a mouse. PrimeScript RT Reagent Kit (Perfect Real Time) was used to synthesize cDNA from RNA. The expression of the ERCC8 gene was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 
Mutant ERCC8 Constructs and Expression in Human Embryonic Kidney 293 Cells
The human embryonic kidney 293 (HEK293) cell line was cultured in a DMEM medium (Corning, Corning, NY, USA) containing 10% fetal bovine serum (FBS; ExCell Bio, China) at 37°C with 5% CO2. The wild type and c.394-398del (p. L132Nfs*6) CDS sequences of the ERCC8 gene were synthesized, subcloned into the pIRES2-EGFP expression vector, and then transfected in 6-well plates using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol. Transfection efficiency was judged by the fluorescence intensity of green fluorescent protein (GFP) after transfection for 48 hours. The cells were then collected to measure the mRNA and protein expression levels of ERCC8 and genes related to transcription-coupled DNA repair (TCR; CSB, XPA, RPA, and XPG), respectively. 
Knockdown of ERCC8 in Human Corneal Fibroblast and Lens Epithelial Cells
The human corneal fibroblast cell line (HTK) and human lens epithelial cells (HLECs) were cultured, as described before.19,24 Specific small interfering RNA (ERCC8 siRNA) was designed and synthesized based on the ERCC8 CDS sequence; it was then transfected in 6-well plates using Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer's protocol. After 48 hours of transfection, the cells were collected to measure the mRNA and protein expression levels of ERCC8, TCR, and matrix metalloproteinase, or collagen Ⅳ, SOD1, respectively. 
To explore the effect of insufficient ERCC8 dosing on the DNA damage repairs of HTK and HLECs, we knocked down ERCC8 in DNA damage cells induced by hydrogen peroxide (H2O2). ERCC8 siRNA was transfected in 6-well or 96-well plates, as described. After 24 hours of transfection, the cells were treated with H2O2 (200 µmol/L) and then collected at 48 hours for subsequent cell viability, apoptosis, and DNA damage and repair-related mRNA and protein level tests. 
Cell Viability, Apoptosis, and DNA Damage Assays
After treatment with H2O2 for 24 hours, cell viability assays of the cells transfected with ERCC8 siRNA and negative controls (NCs) were performed using a Cell Counting Kit-8 (Beyotime, China) according to the manufacturer's protocol. The Hoechst Staining Kit (Beyotime, China) was used to stain the cell nuclear morphism. An Annexin V-FITC Apoptosis Detection Kit (Yeasen, China) was used to detect cell apoptosis levels. The unfolded protein response (UPR)-related genes’ (HSPA5, DDIT3, and ERN1) mRNA expression levels were measured by qRT-PCR. The expression levels of γ- H2AX (a DNA damage marker) were measured with a Western blot. 
mRNA and Protein Expression Levels Measured
Related gene mRNA expression levels in the cell samples were measured with qRT-PCR, as described previously. The primer sequences of the genes used for qRT-PCR are shown in Supplementary Table S1. Related protein expression levels in cell samples were detected by Western blot analyses.19 Primary antibodies included those of collagen IV (ab6586; Abcam), ERCC8 (sc-376981; Santa Cruz), SOD1 (MABC684; Millipore), γ-H2AX (AP0099; Abclonal), NF-kB p65 (phosphor S536, ab76302; Abcam), MMP1 (ab52631; Abcam), Caspase 3 (D320074; Sangon), and GAPDH (KC-5G5; Kangchen, Shanghai, China). 
Results
Clinical Features
The clinical features of the patients in the family are shown in Table 1. The proband (I-2), aged 26 years old, had been diagnosed with congenital cataracts since childhood and had undergone binocular cataract extraction 20 years previously. Seven years ago, her right eye (OD) showed progressive protrusion. She came to the hospital for medical treatment and was diagnosed with KC. Her right eye had nystagmus, corneal ectasia in the central cornea, thinning, scarring in the superficial medium matrix, and a cone-shaped protrusion with Fleischer's ring. The vision of the proband right eye (OD) was 0.05 and could not be corrected. The central corneal thickness of the right eye was 196 µm, and the corneal curvature was 82.75, which were typical KC characteristics (see Table 1). 
Table 1.
 
Clinical Features of the Patients in the Family With Keratoconus and Congenital Cataract
Table 1.
 
Clinical Features of the Patients in the Family With Keratoconus and Congenital Cataract
The clinical examination of her family members showed that her son (II-1) was also a congenital patient with cataract with KC. Lens opacity had been seen in both eyes since birth, and he was diagnosed with congenital cataracts. At the age of 9 years, both of the II-1’s eyes showed abnormal corneal curvature (Ks >49), astigmatism, and uncorrected poor vision, showing the subclinical manifestation of KC (see Table 1). 
WES Analysis Detects a Frameshift Variant in ERCC8
We conducted a WES analysis of the proband and her son to find candidate gene mutations in this family. The average sequencing depth of the WES was 131.00X, and the average coverage was 99.55% (Fig. 1A). With the data analysis and filtering strategy described in the Materials and Methods section, 170 genes with deleterious SNPs/InDels co-segregated with the phenotype of this family were identified, in which 164 genes conform to dominant inheritance, and 6 genes conform to recessive inheritance (Fig. 1B). According to ACMG variant classification guidelines, 38 variants were classified as pathogenic or likely pathogenic (Fig. 1C). Finally, 21 pathogenic or likely pathogenic variants co-segregated with the phenotype in this family were identified (Fig. 1D). Specifically, the ERCC8 gene with the dominant pathogenic frameshift variant has been reported to be associated with congenital cataracts complicated by Cockayne syndrome before.25,26 No pathogenic or likely pathogenic mutations of other reported candidate genes for cataracts27,28 and KC6 were detected. 
Figure 1.
 
Whole exome sequencing (WES) analysis results of patients in a family with keratoconus and congenital cataracts. (A) The sequencing depth and coverage of patients. (B) Variant filtering results. (C) American College of Medical Genetics and Genomics (ACMG) variant-classification results. (D) Twenty-one pathogenic or likely pathogenic variants co-segregated with the phenotype.
Figure 1.
 
Whole exome sequencing (WES) analysis results of patients in a family with keratoconus and congenital cataracts. (A) The sequencing depth and coverage of patients. (B) Variant filtering results. (C) American College of Medical Genetics and Genomics (ACMG) variant-classification results. (D) Twenty-one pathogenic or likely pathogenic variants co-segregated with the phenotype.
The heterozygous frameshift variant in ERCC8 (NM_000082.3: c.394-398del, p. L132Nfs*6) was confirmed with Sanger sequencing. The results showed that the affected members had the heterozygous frameshift variant of ERCC8, whereas the normal member did not (Figs. 2A, 2B). It showed co-segregation with the phenotype of this family. This variant was predicted to be disease-causing by the MutationTaster and was absent from the 1000 Genomes Project databases. The p. L132Nfs*6 change was located in a highly conserved domain (Figs. 2C, 2D) and led to a truncated protein with many protein feature losses (Fig. 2E), which is very likely to affect the normal function of ERCC8. These findings suggested that it was a candidate mutation. 
Figure 2.
 
Pedigree and genetic mutation of the family with keratoconus and congenital cataracts. (A) Pedigree. (B) Sanger sequencing confirms the c.394-398del (p. L132Nfs*6) mutation identified by WES (I-2 and II-1) and HRM (KC5). (C) Cross-species comparison of the region of ERCC8 indicates that the identified mutation affects highly conserved residues. (D) Domain analysis of the p. L132Nfs*6 mutation. (E) The 3D structures of mutant and wild type protein.
Figure 2.
 
Pedigree and genetic mutation of the family with keratoconus and congenital cataracts. (A) Pedigree. (B) Sanger sequencing confirms the c.394-398del (p. L132Nfs*6) mutation identified by WES (I-2 and II-1) and HRM (KC5). (C) Cross-species comparison of the region of ERCC8 indicates that the identified mutation affects highly conserved residues. (D) Domain analysis of the p. L132Nfs*6 mutation. (E) The 3D structures of mutant and wild type protein.
Additional Patients With Sporadic KC With c.394-398del Mutation
To further verify its pathogenicity and examine the frequency of this frameshift mutation in KC and congenital cataracts, it was also genotyped in 262 additional patients with sporadic KC, 20 unrelated congenital patients with cataract, and 210 unrelated healthy controls; one additional patient with KC (KC5) with p. L132Nfs*6 was identified (see Fig. 2B). It was absent in 210 unrelated healthy controls (Table 2). In addition, another disease-causing rare variation (NM_000082.4:c.1080T>C) of ERCC8 was detected in 6 unrelated patients with congenital cataract (see Table 2). These results supported the finding that p. L132Nfs*6 of ERCC8 is associated with KC and congenital cataracts. 
Table 2.
 
Pathogenicity Assessment of ERCC8 Mutation
Table 2.
 
Pathogenicity Assessment of ERCC8 Mutation
ERCC8 Was Expressed Both in the Corneal Stroma and Lens
To determine whether ERCC8 was expressed in the cornea, lens, or other tissues of the eye, we performed ERCC8 protein immunofluorescence staining of mouse eyeballs. The results showed that ERCC8 was highly expressed in the mouse corneal stroma (Fig. 3A). The qRT-PCR results of various tissues of the mouse confirmed that ERCC8 was expressed the most in the cornea, followed by the conjunctiva, lens, lungs, retina, liver, and heart (Fig. 3B). These results suggested that ERCC8 is involved in the normal function of the cornea and lens, and supported the finding that ERCC8 is a disease-causing gene in this family. 
Figure 3.
 
Expression of ERCC8 in different mouse tissues. (A) Representative figures of immunofluorescence staining of ERCC8 in the mouse eye (the upper was a panoramic image, and the lower were local images). (B) The mRNA expression levels of ERCC8 in different mouse tissues.
Figure 3.
 
Expression of ERCC8 in different mouse tissues. (A) Representative figures of immunofluorescence staining of ERCC8 in the mouse eye (the upper was a panoramic image, and the lower were local images). (B) The mRNA expression levels of ERCC8 in different mouse tissues.
The c.394-398del (p. L132Nfs*6) Mutation Leads to an Insufficient Dose of ERCC8 Protein
To examine whether the identified frameshift mutation affected the expression levels of ERCC8, we constructed plasmids with the c.394-398del (p. L132Nfs*6) mutation expressed in HEK293 cells. Immunofluorescence results showed a similar transfection efficiency of the wild type and c.394-398del plasmids (Fig. 4A). Further, qRT-PCR results revealed that c.394-398del (p. L132Nfs*6)-expressing cells showed significantly reduced relative mRNA expression levels of ERCC8 compared with wild type expressing cells (Fig. 4B). The Western blot (Figs. 4C, 4D) also revealed that wild type expressing cells detected significantly higher ERCC8 protein expressions, whereas c.394-398del (p. L132Nfs*6)-expressing cells showed a very weak signal of ERCC8, which is similar to that of cells that are not overexpressed. All these results suggested that the c.394-398del (p. L132Nfs*6) mutation leads to reduced mRNA expression levels and an insufficient dose of ERCC8 protein. 
Figure 4.
 
Mutant ERCC8 constructs and expression in human embryonic kidney 293 cells. (A) The fluorescence intensity of GFP after transfection for 48 hours. (B) mRNA expression levels of ERCC8 and TCR-related genes (CSB, XPA, RPA, and XPG) in overexpressed wild type and mutant cells. (C) Western blot results of ERCC8 protein in overexpressed wild type and mutant cells. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Figure 4.
 
Mutant ERCC8 constructs and expression in human embryonic kidney 293 cells. (A) The fluorescence intensity of GFP after transfection for 48 hours. (B) mRNA expression levels of ERCC8 and TCR-related genes (CSB, XPA, RPA, and XPG) in overexpressed wild type and mutant cells. (C) Western blot results of ERCC8 protein in overexpressed wild type and mutant cells. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Insufficient Dose of ERCC8 Protein Leads to Abnormal Expressions of TCR Genes and Collagen IV in HTK Cells
To examine whether an insufficient dose of ERCC8 protein affected the expression of TCR-related genes and collagen protein in human corneal fibroblast cells (HTK), ERCC8 siRNA was constructed and transfected into the cells. The results showed that ERCC8 siRNA could significantly reduce the mRNA (Fig. 5A) and protein (Figs. 5C, 5D) levels of ERCC8. The ERCC8 siRNA-transfected cells showed significantly decreased expression levels of CSB and XPG and increased expression levels of XPA (see Fig. 5B) compared with the NC cells, which suggested that the insufficient dose of ERCC8 protein caused by ERCC8 siRNA affected transcription-coupled DNA repair in HTK. In addition, the collagen Ⅳ protein level decreased significantly after ERCC8 siRNA transfer (see Figs. 5C, 5D), which suggested an effect of an insufficient dose of ERCC8 protein on the extracellular matrix. There was no significant difference in the expression of SOD1 between ERCC8 siRNA transferred cells and NC cells (see Figs. 5C, 5D). 
Figure 5.
 
Knockdown of ERCC8 in human corneal fibroblast cells. (A) The mRNA expression levels of ERCC8 after being transfected by ERCC8 siRNA. (B) The mRNA expression levels of TCR-related genes (CSB, XPA, RPA, and XPG) after being transfected by ERCC8 siRNA. (C) Western blot results of ERCC8, collagen Ⅳ, and SOD1 proteins after being transfected by ERCC8 siRNA. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Figure 5.
 
Knockdown of ERCC8 in human corneal fibroblast cells. (A) The mRNA expression levels of ERCC8 after being transfected by ERCC8 siRNA. (B) The mRNA expression levels of TCR-related genes (CSB, XPA, RPA, and XPG) after being transfected by ERCC8 siRNA. (C) Western blot results of ERCC8, collagen Ⅳ, and SOD1 proteins after being transfected by ERCC8 siRNA. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Insufficient Dose of ERCC8 Protein Results in the Reduced DNA Damage Repair Ability of HTK Treated by H2O2
To better simulate the DNA damage repair of HTK cells, we induced DNA damage by H2O2 in HTK cells after ERCC8 siRNA transfection. The results showed that ERCC8 siRNA-transfected cells showed significantly decreased cell viability after being treated with H2O2, compared with NC cells (Fig. 6A). The ERCC8 siRNA-transfected cells showed more cells with a rupture in the nuclear membrane and content overflow, indicating more serious damage caused by H2O2 (Fig. 6B). The Western blot (Figs. 6C, 6D) also revealed that the expression levels of γ- H2AX (DNA damage marker), phosphor-p65 (inflammatory marker), and MMP1 (matrix metalloproteinase) were significantly higher in the ERCC8 siRNA-transfected cells compared with the NC cells after H2O2 treatment, which suggested more DNA damage and higher cell inflammatory and matrix metalloproteinase levels in cells with an insufficient dose of ERCC8 protein. 
Figure 6.
 
Cell viability and DNA damage assays of HTK treated by H2O2 after ERCC8 siRNA transfection. (A) Cell viability assay results. (B) Hoechst staining results. (C) Western blot results of ERCC8, γ- H2AX, and phospho p65 protein. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Figure 6.
 
Cell viability and DNA damage assays of HTK treated by H2O2 after ERCC8 siRNA transfection. (A) Cell viability assay results. (B) Hoechst staining results. (C) Western blot results of ERCC8, γ- H2AX, and phospho p65 protein. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Insufficient Dose of ERCC8 Protein Results in Reduced DNA Damage Repair Ability and Increased Apoptosis of HLECs
ERCC8 siRNA-transfected HLECs showed significantly decreased cell viability (Fig. 7A), abnormal TCR-related gene expression (Fig. 7B), and significantly high cell apoptosis levels (Figs. 7C, 7D) after being treated with H2O2, compared with NC cells. The URP-related genes (HSPA5, DDIT3, and ERN1) mRNA expression levels (Fig. 7E) and caspase 3 (apoptotic protease) protein levels (Figs. 7F, 7G) were significantly increased in the ERCC8 siRNA-transfected HLECs, indicating increased UPR-induced apoptosis caused by H2O2. The expression levels of SOD1, γ- H2AX were significantly higher in ERCC8 siRNA-transfected cells, which suggested greater oxidation pressure and DNA damage levels in cells with an insufficient dose of ERCC8 protein. 
Figure 7.
 
Cell viability, apoptosis, and DNA damage assays of HLECs treated by H2O2 after ERCC8 siRNA transfection. (A) Cell viability assay results. (B) The mRNA expression levels of TCR-related genes. (C) Representative figures of immunofluorescence staining of cell apoptosis. (D) Statistical results of the cell apoptosis analysis. (E) The mRNA expression levels of UPR-related genes. (F) Western blot results of SOD1, γ- H2AX, and caspase 3 protein. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Figure 7.
 
Cell viability, apoptosis, and DNA damage assays of HLECs treated by H2O2 after ERCC8 siRNA transfection. (A) Cell viability assay results. (B) The mRNA expression levels of TCR-related genes. (C) Representative figures of immunofluorescence staining of cell apoptosis. (D) Statistical results of the cell apoptosis analysis. (E) The mRNA expression levels of UPR-related genes. (F) Western blot results of SOD1, γ- H2AX, and caspase 3 protein. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Discussion
In this study, using whole exome sequencing of familial cataracts congenital with KC, and screening 262 unrelated KC and 20 unrelated patients with congenital cataract, we identified ERCC8 as a novel gene linked to KC and congenital cataract. A novel frameshift mutation of ERCC8 (c.394-398del, p. L132Nfs*6) was identified in the proband patient and her son from this family. This mutation showed co-segregation with phenotype in this family. It was not found in the 210 unrelated healthy controls and was predicted to alter highly conserved amino acids across the different species and considered to be disease-causing by a function prediction. A screening performed on 262 additional unrelated patients with KC revealed it in one patient. These findings support the idea that p. L132Nfs*6 of ERCC8 is a disease-causing gene mutation in KC. In vitro functional studies provide initial evidence of its role in the pathogenesis of KC and congenital cataracts. 
ERCC8 is located on 5q12.1 and encodes the DNA excision repair protein ERCC-8, a substrate recognition component of the CSA complex that is involved in transcription-coupled nucleotide excision repair.29 This gene is expressed both in the cornea and lens, especially highly in the cornea, as shown in the immunofluorescence staining and qRT-PCR analysis results, which suggest that ERCC8 might play an important role in the maintenance of corneal and lens structure and function via DNA damage repair. The cornea directly exposed to air and light is more susceptible to exogenous oxidizing UV and blue light, which are common DNA damage inducers.30 Previous studies observed increased mitochondrial DNA (mtDNA) damage and double-stranded DNA breaks in KC corneas compared with normal corneas.3133 DNA damage could induce a cell inflammatory response.34 The accumulation of these DNA damage may lead to decreased cell activity and increased cell inflammation levels in the cornea and may result in the loss of normal corneal structure and function. Therefore, DNA damage repair is very important for the cornea. 
The mutant overexpression experiments in this study showed that the c.394-398del (p. L132Nfs*6) mutation of ERCC8 leads to reduced mRNA expression levels and an insufficient dose of the ERCC8 protein. Functional experiments further confirmed that an insufficient dose of the ERCC8 protein leads to decreased expression levels of CSB and XPG and increased expression levels of XPA in HTK. CSB interacts with several transcription and excision repair proteins and may promote complex formation at DNA repair sites.35 XPG is a single-strand-specific DNA endonuclease that is responsible for making the 3′ incision in DNA excision repair.36 XPA is a DNA damage recognition and repair factor and acts as a scaffold to assemble the nucleotide excision repair incision complex at sites of DNA damage.37 The abnormal expression of these TCR genes might lead to the reduced DNA damage repair ability of cells. 
The subsequent experimental results of the HTK DNA damage model again confirmed our inference. The results showed that there was more serious DNA damage in ERCC8 siRNA-transfected HTK cells than in control cells after H2O2 treatment. The insufficient dose of ERCC8 protein results in a reduced DNA damage repair ability of HTK and then leads to decreased cell viability and increased cell inflammation levels of HTK. Inflammation is known to be involved in the pathogenesis of KC, and several studies have reported significantly increased levels of inflammatory markers in KC cells, such as NF-κB, IL-1α, and TNF-α.38,39 Moreover, an insufficient dose of the ERCC8 protein also leads to abnormal expressions of collagens and matrix metalloproteinase, which suggests an effect of an insufficient dose of the ERCC8 protein on the extracellular matrix. Collagens are the main component of the corneal stroma, and matrix metalloproteinases are responsible for proteolytic phenomena. These changes might play an important role in stromal thinning, which is characteristic of KC corneas. Therefore, these functional and cellular changes may affect the normal structure and physiological function of the cornea and play an important role in the pathogenesis of KC. 
At present, there are no reports of KC or other corneal diseases related to ERCC8. However, some patients with congenital cataract condition is complicated by Cockayne syndrome, and they have been reported to be associated with ERCC8 mutations.25,26 ERCC8, also known as CSA, is a well-known candidate gene for Cockayne syndrome (OMIM: 216400) and UV-sensitive syndrome 2 (OMIM: 614621) with autosomal recessive inheritance. Some patients with Cockayne syndrome had ocular manifestations of congenital cataract.25,26 Our familial patients, only harboring heterozygous mutations of ERCC8, did not show other known symptoms of Cockayne syndrome except for congenital cataracts. Identification of another disease-causing rare variation (c.1080T>C) of ERCC8 in 6 unrelated patients with congenital cataract supports that ERCC8 is associated with congenital cataracts. The experimental results of the DNA damage model also showed that the insufficient dose of ERCC8 protein results in the reduced DNA damage repair ability of HLECs. This then leads to decreased cell viability and increased cell apoptosis levels in HLECs via aberrant activation of the unfolded protein response. An unfolded protein response is known to be involved in the pathogenesis of cataract, and several studies have reported that it is involved in cataract formation.4043 Cockayne syndrome-associated CSA mutations were reported to impair protein folding before.26 All these findings support the idea that these functional and cellular changes caused by an insufficient dose of ERCC8 protein play an important role in the pathogenesis of cataracts. 
Acknowledgments
Supported by the Shandong Provincial Natural Science Foundation, China (ZR2020MC059 and ZR2021MH074), the National Natural Science Foundation of China (82101164), and the China Postdoctoral Science Foundation (2019M652311). 
Disclosure: X.-D. Hao, None; Y.-Z. Yao, None; K.-G. Xu, None; B. Dong, None; W.-H. Xu, None; J.-J. Zhang, None 
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Figure 1.
 
Whole exome sequencing (WES) analysis results of patients in a family with keratoconus and congenital cataracts. (A) The sequencing depth and coverage of patients. (B) Variant filtering results. (C) American College of Medical Genetics and Genomics (ACMG) variant-classification results. (D) Twenty-one pathogenic or likely pathogenic variants co-segregated with the phenotype.
Figure 1.
 
Whole exome sequencing (WES) analysis results of patients in a family with keratoconus and congenital cataracts. (A) The sequencing depth and coverage of patients. (B) Variant filtering results. (C) American College of Medical Genetics and Genomics (ACMG) variant-classification results. (D) Twenty-one pathogenic or likely pathogenic variants co-segregated with the phenotype.
Figure 2.
 
Pedigree and genetic mutation of the family with keratoconus and congenital cataracts. (A) Pedigree. (B) Sanger sequencing confirms the c.394-398del (p. L132Nfs*6) mutation identified by WES (I-2 and II-1) and HRM (KC5). (C) Cross-species comparison of the region of ERCC8 indicates that the identified mutation affects highly conserved residues. (D) Domain analysis of the p. L132Nfs*6 mutation. (E) The 3D structures of mutant and wild type protein.
Figure 2.
 
Pedigree and genetic mutation of the family with keratoconus and congenital cataracts. (A) Pedigree. (B) Sanger sequencing confirms the c.394-398del (p. L132Nfs*6) mutation identified by WES (I-2 and II-1) and HRM (KC5). (C) Cross-species comparison of the region of ERCC8 indicates that the identified mutation affects highly conserved residues. (D) Domain analysis of the p. L132Nfs*6 mutation. (E) The 3D structures of mutant and wild type protein.
Figure 3.
 
Expression of ERCC8 in different mouse tissues. (A) Representative figures of immunofluorescence staining of ERCC8 in the mouse eye (the upper was a panoramic image, and the lower were local images). (B) The mRNA expression levels of ERCC8 in different mouse tissues.
Figure 3.
 
Expression of ERCC8 in different mouse tissues. (A) Representative figures of immunofluorescence staining of ERCC8 in the mouse eye (the upper was a panoramic image, and the lower were local images). (B) The mRNA expression levels of ERCC8 in different mouse tissues.
Figure 4.
 
Mutant ERCC8 constructs and expression in human embryonic kidney 293 cells. (A) The fluorescence intensity of GFP after transfection for 48 hours. (B) mRNA expression levels of ERCC8 and TCR-related genes (CSB, XPA, RPA, and XPG) in overexpressed wild type and mutant cells. (C) Western blot results of ERCC8 protein in overexpressed wild type and mutant cells. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Figure 4.
 
Mutant ERCC8 constructs and expression in human embryonic kidney 293 cells. (A) The fluorescence intensity of GFP after transfection for 48 hours. (B) mRNA expression levels of ERCC8 and TCR-related genes (CSB, XPA, RPA, and XPG) in overexpressed wild type and mutant cells. (C) Western blot results of ERCC8 protein in overexpressed wild type and mutant cells. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Figure 5.
 
Knockdown of ERCC8 in human corneal fibroblast cells. (A) The mRNA expression levels of ERCC8 after being transfected by ERCC8 siRNA. (B) The mRNA expression levels of TCR-related genes (CSB, XPA, RPA, and XPG) after being transfected by ERCC8 siRNA. (C) Western blot results of ERCC8, collagen Ⅳ, and SOD1 proteins after being transfected by ERCC8 siRNA. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Figure 5.
 
Knockdown of ERCC8 in human corneal fibroblast cells. (A) The mRNA expression levels of ERCC8 after being transfected by ERCC8 siRNA. (B) The mRNA expression levels of TCR-related genes (CSB, XPA, RPA, and XPG) after being transfected by ERCC8 siRNA. (C) Western blot results of ERCC8, collagen Ⅳ, and SOD1 proteins after being transfected by ERCC8 siRNA. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Figure 6.
 
Cell viability and DNA damage assays of HTK treated by H2O2 after ERCC8 siRNA transfection. (A) Cell viability assay results. (B) Hoechst staining results. (C) Western blot results of ERCC8, γ- H2AX, and phospho p65 protein. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Figure 6.
 
Cell viability and DNA damage assays of HTK treated by H2O2 after ERCC8 siRNA transfection. (A) Cell viability assay results. (B) Hoechst staining results. (C) Western blot results of ERCC8, γ- H2AX, and phospho p65 protein. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Figure 7.
 
Cell viability, apoptosis, and DNA damage assays of HLECs treated by H2O2 after ERCC8 siRNA transfection. (A) Cell viability assay results. (B) The mRNA expression levels of TCR-related genes. (C) Representative figures of immunofluorescence staining of cell apoptosis. (D) Statistical results of the cell apoptosis analysis. (E) The mRNA expression levels of UPR-related genes. (F) Western blot results of SOD1, γ- H2AX, and caspase 3 protein. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Figure 7.
 
Cell viability, apoptosis, and DNA damage assays of HLECs treated by H2O2 after ERCC8 siRNA transfection. (A) Cell viability assay results. (B) The mRNA expression levels of TCR-related genes. (C) Representative figures of immunofluorescence staining of cell apoptosis. (D) Statistical results of the cell apoptosis analysis. (E) The mRNA expression levels of UPR-related genes. (F) Western blot results of SOD1, γ- H2AX, and caspase 3 protein. (D) Statistical results of the Western blot; N = 3, *, P < 0.05.
Table 1.
 
Clinical Features of the Patients in the Family With Keratoconus and Congenital Cataract
Table 1.
 
Clinical Features of the Patients in the Family With Keratoconus and Congenital Cataract
Table 2.
 
Pathogenicity Assessment of ERCC8 Mutation
Table 2.
 
Pathogenicity Assessment of ERCC8 Mutation
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