## 4 Discussion
This alteration could have consequences, such as exon skipping, for example, leading to the exclusion of an entire exon from _VSX1_ pre-mRNA transcript and consequently affecting gene expression. However, these findings need further investigation and validation through in vitro functional analysis. MiR-203 is a widely studied miRNA for mediation in repressing several types of disorders, including multiple types of malignancies and cancer [38; 39]. miRNA-203 has been found to suppress cell proliferation and the metastasis of several types of colorectal cancers by targeting Eukaryotic initiation factor 5A2 (EIF5A2) [40]. On the other hand, miRNA-3938 not only plays a role in cancers but has also been reported to affect macrophagecell development [41]. The loss of binding of miRNA-203 with _VSX1 3_UTR c*496A>G (MT) and the binding of miRNA-3938 could be implicated in KC; however, the presence of variant c*496A>G heterozygosity in KC patients could indicate a penetrance/dosage effect because one of the parents that carried the variation did not exhibit the phenotype, whereas disease manifestation was seen in offspring. The dosage effect of _VSX1_ variants in KC families has been observed in one previous study [42], which reported the Q175H _VSX1_ variant as being pathogenic with incomplete penetrance in the patient but not in the carrier mother. The genetic variants demonstrating such reduced penetrance have been reported to involve some modifier genes or environmental factors for disease presentation. It is also possible that the variant c*496A>G requires some genetic modifier or environmental factor to be pathogenic. Like the previous studies by [PERSON] et al. [28], [PERSON] et al. [29], [PERSON] et al. [30], [PERSON] et al. [31], [PERSON] et al. [32] and [PERSON] et al. [33] on _VSX1_ screening in different ethnicities worldwide (Table 5), there seems a similarity in previous studies regarding the cohort selection and sample size. However, none of the studies were able to solve a complete cohort involving sporadic as well as familial cases. Most of the studies reported polymorphic variations, including synonymous as well as non-synonymous and intronic variations; however, due to the absence of in silico analysis, the exact role of the non-coding variations and microRNA was not investigated in previous studies. These contradictory results, therefore, highlight and suggest that KC cohorts, including sporadic and familial cases belonging to different ethnicities, to be genetically screened using state-of-the-art techniques, such as genome and exome sequencing to identify the exact genetic cause. These limitations in the current and previous studies, therefore, suggest thorough investigations involving larger KC cohorts and genome sequencing of KC patients. Another limitation of the current and previous findings is the lack of in vitro and in vivo functional characterization of identified genetic variants in KC patients. Regarding the cis-acting element and their predicted association with microRNA in the current study, though functional analysis could not be performed, however number of studies have reported the role of microRNA-203 in the increased inflammatory response [43]. The role of inflammation in KC pathogenesis is well defined [44; 45]; therefore, the functional validation of the role of microRNAs in KC manifestation is suggested in future KC investigations. \begin{table}
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