## 4 Discussion  
This study was designed to investigate the contribution of _VSX1_ in autosomal recessive genes in Pakistani keratoconus families compared with that in subjects appearing as sporadic cases. This study was also set to perform an in silico analysis of the rare variants to allow for the identification of cis-acting elements in _VSX1_. In the present study, screening of the previously reported KC-associated gene _VSX1_ revealed a potential role for rare variants which might contribute to KC manifestation. This was achieved through the identification of novel cis-acting elements in _VSX1_ based on bioinformatic analysis of the gene. The results of the _VSX1_ screening in the current panel were not in agreement with those reported earlier, where most of the cases carried mutations in the coding part of the gene and were reported for the dominant form of the disease. However, in the present cohort of autosomal recessive KC families, screening of _VSX1_ found no novel or recurrent mutation except for two SNPs rs12480307, a synonymous variation, and a 3'UTR rs76499395. A reasonable number of studies using _VSX1_ mutational screening reported _VSX1_ exon two as a mutation hot spot in KC cases, but the SNPs observed in two patients in the present study lie in the third exon and 3'UTR sequences. The results of the sporadic cases were also like that of the familial probands, exhibiting no _VSX1_ mutations. Regarding the mutation identification, the findings of the present study were consistent with those undertaken in Caucasian [28], Czech [29], Italian [30], Slovenian [31], South Indian [32] and North Indian [33] populations, in which no coding sequence _VSX1_ mutations were found in KC cases while ignoring very rare variants and their possible role in disease manifestation. Moreover, the expression analysis of _VSX1_ in human and mouse corneas also indicated contradictory findings. In humans [19], _VSX1_ expression in the adult cornea was not detected, but it was found to be higher in retinal tissues [34]. Although polymorphisms in different genes have been reported to be one of the risk factors in the pathogenesis of KC worldwide, a number of studies have reported that the association of SNPs with KC is low but did not investigate the underlying gene regulatory mechanisms, SREs and miRNAs which might be implicated in such associations. The identification of the ESE element (containing c.546A>G SNP) and a miRNA binding site at _VSX1_ 3'UTR c.*496 position has highlighted the functional aspects of SNPs on cis-acting elements and their consequent effect on gene expression. Among the regulatory sequences, splicing is determined by a regulatory complex comprising a number of snRNP (small nuclear ribonuclete proteins) often named after their small nuclear RNA (snRNA) segments U1, U2, U4, U5 and U6 [35]. The role of snRNPs is to identify and bind at specific splice donor, branch and acceptor sites characterized by GU, polypyrrimidine and AG nucleotides lying in the intronic region. The recruitment of these proteins is initiated by specific serine/arginine-rich (SR) proteins, the SRSF or SR (specific serine/arginine-rich) proteins. These start the recruitment process of snRNPs only after binding at specific exonic splicing enhancer (ESE) motifs lying in the flanking exonic regions of the subject introns. Twelve different SRSF proteins are known to bind at their specific ESE elements in pre-mRNA to initiate the splicing regulatory complex. Once the complex is formed, the spliceosome splices out the intronic region joining the two neighboring exons to form the mature mRNA for translation [36; 37]. The current findings, therefore, clearly indicated that the _VSX1_ c546-552 was occupied by a highly putative ESE site, and in the case of the SNP c.546A>G, the homozygously present G allele, the ESE loses its affinity for a specific SR protein.