## 3 Results
### In Silico Analysis of c.546A>G  
The rs12480307 (c.546A>G; p. Ala182 Ala) sequence lies in the third exon of the _VSX1_, which is close to a splice site; therefore, the SNP, along with flanking sequences, was analyzed to determine whether these contained any ESE or ESS sequences. It was observed that the _VSX1_ SNP c.546A>G was altered and broke an ESE element lying in the third exon of the _VSX1_. This has been observed using three different splicing analytical tools used in this study. HSF predicted an ESE element starting from position _VSX1_ c.546, spanning over  
Figure 2: Sequence chromatogram of observed variants. Upper panel (**A**) represents normal and mutated sequence for rs12480307 (c.546A>G) while lower panel (**B**) is of rs76499395 (c.*496A>G) polymorphism. the next seven 3\({}^{\prime}\) nucleotides with the sequence ATTCAGC. HSF predicted that in terms of SNP c.546A>G, the ESE element would be altered and broken. RESCUE-ESE also predicted the same ESE at position c546-552 of _VSX1_. The EIE identifier also predicted an exonic SRE at the same site (Table 2). The ESE finder identified a strong SRSF5 binding motif ATTCAGC (_VSX1_ c546-552) with a high binding score of 3.07, well above the threshold score of 2.67. In the case of SNP c.546A>G SRSF5, binding was lost for SRSF 5, but the mutant motif GTTCAGC showed a binding affinity with the SRSF2 protein (Table 3). Figure 3 illustrates the pre-mRNA analogs of the ESE elements, _VSX1_ Wild Type c.546A-c.553G (ATTCAGCC) and _VSX1_ SNP c.546A>G-c.553G (GTTCAGCG), bound to SRSF5 as revealed by docking. The wild type of ESE segment, c.546A-c.553G (ATTCAGCG), binds right in the binding pocket of the SRSF 5 protein, whereas the SNP segment, c.546A>G-c.553G (GTTCAGCG), did not bind well except for its initial 5\({}^{\prime}\) region, docking nearly 3-4 A away from the binding pocket of the SRSF 5 molecule. This would affect the splicing of the corresponding exonic region. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Predicted Signal** & **Prediction Algorithm** & **cDNA Position** & **Interpretation** \\ \hline \multirow{4}{*}{ESE Site Broken} & 1. ESE-Finder-SRp40 & \multirow{4}{*}{\begin{tabular}{} \end{tabular} } & \multirow{4}{*}{\begin{tabular}{} \end{tabular} } \\ \cline{2-2} \cline{4-4}  & 2. RESCUE ESE Hexamers & & & \\ \cline{2-2} \cline{4-4}  & 3. EIEs from [PERSON] et al. [23] & \multirow{2}{*}{\
\begin{tabular}{} \end{tabular} } & \multirow{2}{*}{
\begin{tabular}{} \end{tabular} } \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Splicing analysis of the exon 3 sequence of the _VSX1_ and its SNP c.546A>G, effect on the ESE site the formation of splicing regulatory complex. Figure 3: ESE elements, _VSX1_ Wild Type c.546A-c.553G (ATTCAGCG) shown in brown and _VSX1_ SNP c.546A>G-c.553G (GTTCAGCG) showing green docked to the SRSF 5 binding pocket. Notice the Wild Type ESE element binds well into the SRSF 5 binding pocket, while the green one binds away, excepting its 5\({}^{\prime}\) region. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Type** & \multicolumn{4}{c}{**SRSF Binding Properties**} \\ \cline{2-5}  & **Biding Motif** & **SRSF Protein** & **Binding Score** & **BS Threshold** \\ \hline _VSX1_ Wild Type & c.546A-c.553G & ATTCAGCG & SRSF5 & 3.07 & 2.67 \\ \hline _VSX1_ SNP & c.546A>G-c.553G & GTTCAGCG & SRSF2 & 3.20 & 2.38 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Variation in ESE-SRSF protein binding properties between _VSX1_ Wild Type c.546A as compared to _VSX1_ SNP c.546A>G-c.553G analyzed using the ESE finder. Both PITA and miRANDA predicted a miRNA binding site at _VSX1_ 3'UTR c.*496 position (Table 4). The wildtype (WT) sequence was found to show strong binding with miRNA hsa-miR-203, and the binding site spanned across c*475 to c.*496 in _VSX1_ 3'UTR wildtype (_VSX1_ 3'UTR (WT) (Figure 4A). PITA estimated \(\Delta\)\(\Delta\)G values for miRNA-203 binding with _VSX1_ 3'UTR (WT) as \(-\)9.34, the seed region spanned over eight base pairs with all bases pairing up with target mRNA as one wobble pair.