Prediction and assessment of deleterious and disease causing nonsynonymous single nucleotide polymorphisms (nsSNPs) in human FOXP4 gene: An in - silico study

Abstract

In humans, FOXP gene family is involved in embryonic development and cancer progression. The FOXP4 (Forkhead box protein P4) gene belongs to this FOXP gene family. FOXP4 gene plays a crucial role in oncogenesis. Single nucleotide polymorphisms are biological markers and common determinants of human diseases. Mutations can largely affect the function of the corresponding protein. Therefore, the molecular mechanism of nsSNPs in the FOXP4 gene needs to be elucidated. Initially, the SNPs of the FOXP4 gene were extracted from the dbSNP database and a total of 23124 SNPs was found, where 555 nonsynonymous, 20525 intronic, 1114 noncoding transcript, 334 synonymous were obtained and the rest were unspecified. Then, a series of bioinformatics tools (SIFT, PolyPhen2, SNAP2, PhD SNP, PANTHER, I-Mutant2.0, MUpro, GOR IV, ConSurf, NetSurfP 2.0, HOPE, DynaMut2, GeneMANIA, STRING and Schrodinger) were used to explore the effect of nsSNPs on FOXP4 protein function and structural stability. First, 555 nsSNPs were analyzed using SIFT, of which 57 were found as deleterious. Following, PolyPhen2, SNAP2, PhD SNP and PANTHER analyses, 10 nsSNPs (rs372762294, rs141899153, rs142575732, rs376938850, rs367607523, rs112517943, rs140387832, rs373949416, rs373949416 and rs376160648) were common and observed as deleterious, damaging and diseases associated. Following that, using I-Mutant2.0 and MUpro servers, 7 nsSNPs were found to be the most unstable. GOR IV predicted that these seven nsSNPs affect protein structure by altering the protein contents of alpha helixes, extended strands, and random coils. Following DynaMut2, 5 nsSNPs showed a decrease in the ΔΔG value compared with the wild-type and were found to be responsible for destabilizing the corresponding protein. GeneMANIA and STRING network revealed interaction of FOXP4 with other genes. Finally, molecular dynamics simulation analysis revealed consistent fluctuation in RMSD and RMSF values, Rg and hydrogen bonds in the mutant proteins compared with WT, which might alter the functional and structural stability of the corresponding protein. As a result, the aforementioned integrated comprehensive bioinformatic analyses provide insight into how various nsSNPs of the FOXP4 gene change the structural and functional properties of the corresponding protein, potentially proceeding with the pathophysiology of human diseases.

Keywords: FOXP4 gene; Functional prediction; Secondary and tertiary structure and interaction analysis; Stability prediction.

Fig. 1

The complete workflow of nsSNPs analysis.

Fig. 2

(A) Prediction of evolutionary conserved amino acid residues by ConSurf server Conservation score is represented as the color coding bars. (B) Surface accessibility prediction of nsSNPs of the FOXP4 protein by NetSurfP 2.0. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Interaction of FOXP4 with other genes and proteins. (A) Gene-gene interaction network of FOXP4 gene predicted by GeneMANIA. (B) Protein-protein interaction of FOXP4 gene by STRING tool.

Fig. 4

Results of 50 ns molecular dynamics simulation by Schrodinger. (A) RMSD values of native and mutant structures. (B) RMSF values of native and mutant structures. (C) Calculation of Rg over the entire simulation. (D) Calculation of H-bonds represented as a time-depend. The color scheme is as follows: native (blue), Q156H mutant (red), T330I mutant (green), Q355H mutant (violet), R373W mutant (yellow), and L679P mutant (orange). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)