Predicting the most deleterious missense nsSNPs of the protein isoforms of the human HLA-G gene and in silico evaluation of their structural and functional consequences

Abstract

Background: The Human Leukocyte Antigen G (HLA-G) protein is an immune tolerogenic molecule with 7 isoforms. The change of expression level and some polymorphisms of the HLA-G gene are involved in various pathologies. Therefore, this study aimed to predict the most deleterious missense non-synonymous single nucleotide polymorphisms (nsSNPs) in HLA-G isoforms via in silico analyses and to examine structural and functional effects of the predicted nsSNPs on HLA-G isoforms.

Results: Out of 301 reported SNPs in dbSNP, 35 missense SNPs in isoform 1, 35 missense SNPs in isoform 5, 8 missense SNPs in all membrane-bound HLA-G isoforms and 8 missense SNPs in all soluble HLA-G isoforms were predicted as deleterious by all eight servers (SIFT, PROVEAN, PolyPhen-2, I-Mutant 3.0, SNPs&GO, PhD-SNP, SNAP2, and MUpro). The Structural and functional effects of the predicted nsSNPs on HLA-G isoforms were determined by MutPred2 and HOPE servers, respectively. Consurf analyses showed that the majority of the predicted nsSNPs occur in conserved sites. I-TASSER and Chimera were used for modeling of the predicted nsSNPs. rs182801644 and rs771111444 were related to creating functional patterns in 5'UTR. 5 SNPs in 3'UTR of the HLA-G gene were predicted to affect the miRNA target sites. Kaplan-Meier analysis showed the HLA-G deregulation can serve as a prognostic marker for some cancers.

Conclusions: The implementation of in silico SNP prioritization methods provides a great framework for the recognition of functional SNPs. The results obtained from the current study would be called laboratory investigations.

Keywords: Deleterious SNPs; HLA-G gene; In silico analysis; Missense mutation; Structural and functional impact.

Fig. 1

HLA-G heavy chain gene comprises 7 introns (i1-i7) and 8 exons (each with a distinctive color) with an internal stop codon in Exon 6. As shown in figure each exon encodes a discrete part of the heavy chain, except exon 7 and 8. Alternative splicing events of HLA-G primary transcript (exclusion exon 3 or/and exon 4 and keeping of intron 4 or intron 2 from the final gene transcript) generate seven isoforms. Soluble isoforms lack the transmembrane and cytoplasmic regions due to the intron retention, which includes an immature stop codon. HLA-G5 and HLA-G6 have a tail (21 amino acids) that plays a role in their solubility. HLA-G1 is the complete molecule. HLAG1 is homologous to HLA-G5 and both of them associate with B2M. The signal peptide (exon 1) and α1 domain (exon 2) are existing in all isoforms. Figure modified from Bainbridge et al. [14]

Fig. 2

Flowchart of the different steps of the study

Fig. 3

The 3-D pie-chart shows the percentage of missense SNPs, 5′ UTR, 3′ UTR and other types of SNPs in HLA-G gene (according to the dbSNP database on December 2018)

Fig. 4

Consurf analysis of HLA-G1. The degree of conservation of amino acids was shown in the colouring scheme. The color intensity increases based on amino acids conservation grades e.g. turquoise indicates variable sites; white indicates average sites; maroon indicates evolutionarily conserved sites. The most deleterious predicted SNPs are marked below the sequence as red arrows. e is the exposed residue. b is the buried residue. f is an estimated functional residue (highly conserved and exposed). s is an estimated structural residue (highly conserved and buried)

Fig. 5

Protein–protein interaction network of HLAG with 10 partners

Fig. 6

The correlation of deregulation of HLA-G gene and overall survival rate of the cancer patients was evaluated using Kaplan-Meier plotter