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.
31–33 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 H
2O
2 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.
40–43 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.