In the present study, nine novel mutations were identified in FNDC3B (c.455C > T:p.P152L),COL4A4 ( c.3636_3637del:p.R1212fs),MPDZ (c.5015G > T:p.R1672L; c.3798dupA:p.P1267fs; c.28G > A:p.A10T), DOCK9 (c.1940C > T:p.P647L), POLG (c.127_128insGGC:p.Q43delinsRQ), IPO5 (c.3019G > A:p.V1007I), and TGFBI (c.624 + 7->A) in eight of the fifty-two patients with KC of Han Chinese ethnicity. The mutation in FNDC3B (c.455C > T:p.P152L) coexisted with the mutation in COL4A4 ( c.3636_3637del:p.R1212fs) in one female KC patient (Table 3), and the mutation DOCK9 (c.1940C > T:p.P647L) coexisted with a potentially damaging ZNF469( (c.3466G > A ) mutation in one male KC patient. All of the mutations were found in sporadic KC cases and were absent in the results of the whole-exome sequencing (WES) data acquired. None of the nine mutations related to KC have been previously reported.
Since the first case of KC was confirmed in 1854 , numerous studies were conducted to uncover the potential pathophysiology. It should be noted that positive family history was found in 6–20% of KC cases , and concordance between monozygotic twins was confirmed compared with monozygotic twins, which suggest that genetic factors are dominant components in KC etiology.
As is well known, the human cornea is rich in extracellular matrix (ECM), which maintains corneal transparency and biomechanical strength . Through medical biochemistry techniques—such as immunostaining technology—and proteomics, different abnormalities related to varying risk genes in the expression of ECM components were identified in KC patients . To date, more than ten KC-related genes have been identified as related to ECM component abnormality [13–16]. In the present study, two KC-related mutations were identified in two well-known ECM-associated genes (TGFBI and COL4A4). In KC patients, Bykhovskaya et al. identified a significantly unregulated transcript of the gene TGFBI, which was reported as related to the down-regulation of collagen genes (such as COL5A1, coding for collagen V) in ECM and caused decreased CCT . TGFBI encodes the TGFβ-induced protein and works as a transcription regulator; this extracellular protein can also mediate cell adhesion to collagen, laminin, fibronectin, and proteoglycans . In this study, the coding region of TGFBI was screened, and one novel TGFBI mutation (c.624 + 7->A) was identified, which further confirmed that TGFBI might be a risk gene for KC patients in China. Collagens are the main components of ECM, and the type IV collagen family (COL4A1 to COL4A6) consists of the corneal basement membrane. The COL4A4 gene was expressed by the central corneal epithelium. With respect to the function of collagen IV, COL4A4 was also suggested to be a candidate risk gene for KC [19–20]. In this study, one COL4A4 ( c.3636_3637del:p.R1212fs) that has never been reported in Chinese KC patients was identified.
Recent studies show that part of the genes involved in KC development were related with CCT. In the present study, FNDC3B and MPDZ are widely thought of as CCT-related genes, and progressive corneal thinning is known to be a feature of the pathophysiology of KC. Lu et al. reported that rs4894535 in FNDC3B and rs1324183 in MPDZ led to decreased CCT with GWAS , and the results were replicated in studies by Hao and Sahebjada [22–23].In the present study, one novel FNDC3B (c.455C > T:p.P152L) mutation and three MPDZ (c.5015G > T:p.R1672L; c.3798dupA:p.P1267fs; c.28G > A:p.A10T) mutations were identified in Chinese KC patients; this was consistent with former studies and provided strong genetic evidence that variants in these two genes lead to increased susceptibility to KC. Nevertheless, the role of FNDC3B and MPDZ in KC remains elusive.
DOCK9 variants were first indicated to be related to KC risk in an Ecuadorian family . Former fundamental studies have shown that DOCK9 could work as an activator of the cell division cycle and regulate the would repairing process in the human cornea . With next-generation sequencing technology, this study’s KC cohort found one novel mutation (c.1940C > T:p.P647L) that was located in the exon region in DOCK9, and the mutation was predicted to be damaging by SIFT.
IPO5, which encodes a member of the kariopherin super-family and is involved in protein nuclear transport[25–26], was also screened in our study. One novel mutation (c.3019G > A:p.V1007I) was indicated to be related to KC in this Chinese KC cohort. Consistent with us, Justyna et al. also reported IPO5 mutations in Polish KC patients .
We also screen the coding region of POLG, which encodes the only DNA polymerase present in mammalian mitochondria [28–29]. Wojcik et al. provided strong evidence that the POLG gene might play a vital role in KC pathogenesis and determining the risk of this corneal disease .However, genetics studies about the relationship between KC and POLG are limited. The results of this study provided new evidence that supports Wojcik’s conclusion.
This study had some limitations. First, the limited sample size influenced the significance of the present study. Secondly, just the coding region of the identified genes was screened, while deep intronic substitutions could also induce KC formation. Thirdly, negative family history indicated that all involved patients were sporadic. Thus, it is difficult to define the genetic pattern of novel mutations. In our view, the absence of these mutations in the majority of Chinses KC patients suggests that its role is not dominant, and the phenotype of the next generation would help to define the genetic pattern. Further researches should be designed to uncover the potential role of the novel mutations in KC etiology by analysis of the corneal characteristics of genetic manipulation in animal models and corneal materials obtained from keratoplasty surgeries of mutations carriers.