RESULTS: Positively significant departures from neutral expectations were detected on the surf4.1region encoding C-terminus of the variable region 2 (Var2) by 3 population-based tests in the western Kenyan population as similar in the Thai population, which was not covered by the previous analysis for eastern Kenyan population. Significant excess of non-synonymous substitutions per nonsynonymous site over synonymous substitutions per synonymous site was also detected in the Var2 region. Negatively significant departures from neutral expectations was detected on the region encoding Var1 C-terminus consistent to the previous observation in the eastern Kenyan population. Parasites possessing a frameshift mutation resulting a product without intracellular Trp-rich (WR) domains were 22/23 in western Kenya and 22/36 in Thailand. More than one copy of surf4.1gene was detected in western Kenya (4/24), but no CNV was found in Thailand (0/36).
CONCLUSIONS: The authors infer that the high polymorphism of SURFIN4.1Var2 C-terminus in both Kenyan and Thai populations were shaped-up by diversifying selection and maintained by balancing selection. These phenomena were most likely driven by immunological pressure. Whereas the SURFIN4.1Var1 C-terminus is suggested to be under directional selection consistent to the previous report for the eastern Kenyan population. Most western Kenyan isolates possess a frameshift mutation that would limit the expression of SURFIN4.1on the merozoite, but only 60% of Thai isolates possess this frameshift, which would affect the level and type of the selection pressure against this protein as seen in the two extremities of Tajima's D values for Var1 C-terminus between Kenyan and Thai populations. CNV observed in Kenyan isolates may be a consequence of this frameshift mutation to increase benefits on the merozoite surface.
MATERIAL AND METHODS: All the information for CYP1B1 missense variants was retrieved from the dbSNP database. Seven different tools, namely: SIFT, PolyPhen-2, PROVEAN, SNAP2, PANTHER, PhD-SNP, and Predict-SNP, were used for functional annotation, and two packages, which were I-Mutant 2.0 and MUpro, were used to predict the effect of the variants on protein stability. A phylogenetic conservation analysis using deleterious variants was performed by the ConSurf server. The 3D structures of the wild-type and mutants were generated using the I-TASSER tool, and a 50 ns molecular dynamic simulation (MDS) was executed using the GROMACS webserver to determine the stability of mutants compared to the native protein. Co-expression, protein-protein interaction (PPI), gene ontology (GO), and pathway analyses were additionally performed for the CYP1B1 in-depth study.
RESULTS: All the retrieved data from the dbSNP database was subjected to functional, structural, and phylogenetic analysis. From the conducted analyses, a total of 19 high-risk variants (P52L, G61E, G90R, P118L, E173K, D291G, Y349D, G365W, G365R, R368H, R368C, D374N, N423Y, D430E, P442A, R444Q, F445L, R469W, and C470Y) were screened out that were considered to be deleterious to the CYP1B1 gene. The phylogenetic analysis revealed that the majority of the variants occurred in highly conserved regions. The MD simulation analysis exhibited that all mutants' average root mean square deviation (RMSD) values were higher compared to the wild-type protein, which could potentially cause CYP1B1 protein dysfunction, leading to the severity of the disease. Moreover, it has been discovered that CYP1A1, VCAN, HSD17B1, HSD17B2, and AKR1C3 are highly co-expressed and interact with CYP1B1. Besides, the CYP1B1 protein is primarily involved in the metabolism of xenobiotics, chemical carcinogenesis, the retinal metabolic process, and steroid hormone biosynthesis pathways, demonstrating its multifaceted and important roles.
DISCUSSION: This is the first comprehensive study that adds essential information to the ongoing efforts to understand the crucial role of genetic signatures in the development of PCG and will be useful for more targeted gene-disease association studies.