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In-Silico Investigation of Effects of Single-Nucleotide Polymorphisms in PCOS-Associated CYP11A1 Gene on Mutated Proteins.

ABSTRACT: Polycystic ovary syndrome (PCOS) is a reproductive disorder with multiple etiologies, mainly characterized by the excess production of androgens. It is equally contributed to by genes and environment. The CYP11A1 gene is imperative for steroidogenesis, so any dysregulation or mutation in this gene can lead to PCOS pathogenesis. Therefore, nucleotide diversity in this gene can be helpful in spotting the likelihood of developing PCOS. The present study was initiated to investigate the effect of single nucleotide polymorphisms in human CYP11A1 gene on different attributes of encoded mutated proteins, i.e., sub-cellular localization, ontology, half-life, isoelectric point, instability index, aliphatic index, extinction coefficient, 3-D and 2-D structures, and transmembrane topology. For this purpose, initially coding sequence (CDS) and single nucleotide polymorphisms (SNPs) were retrieved for the desired gene from Ensembl followed by translation of CDS using EXPASY tool. The protein sequence obtained was subjected to different tools including CELLO2GO, ProtParam, PHYRE2, I-Mutant, SIFT, and PolyPhen. It was found that out of seventy-eight SNPs analyzed in this project, seventeen mutations, i.e., rs750026801 in exon 1, rs776056840, rs779154292 and rs1217014229 in exon 2, rs549043326 in exon 3, rs755186597 in exon 4, rs1224774813, rs757299093 and rs1555425667 in exon 5, rs1454328072 in exon 7, rs762412759 and rs755975808 in exon 8, and rs754610565, rs779413653, rs765916701, rs1368450780, and rs747901197 in exon 9 considerably altered the structure, sub-cellular localization, and physicochemical characteristics of mutated proteins. Among the fifty-nine missense SNPs documented in present study, fifty-five and fifty-three were found to be deleterious according to SIFT and PolyPhen tools, respectively. Forty-nine missense mutations were analyzed to have a decreasing effect on the stability of mutant proteins. Hence, these genetic variants can serve as potential biomarkers in human females for determining the probability of being predisposed to PCOS.

SUBMITTER: Muccee F

PROVIDER: S-EPMC9317558 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The advent of high throughput sequencing technology has enabled the 1000 Genomes Project Pilot 3 to generate complete sequence data for more than 906 genes and 8,140 exons representing 697 subjects. The 1000 Genomes database provides a critical opportunity for further interpreting disease associations with single nucleotide polymorphisms (SNPs) discovered from genetic association studies. Currently, direct sequencing of candidate genes or regions on a large number of subjects remains both cost- and time-prohibitive.To accelerate the translation from discovery to functional studies, we propose an in silico gene sequencing method (ISS), which predicts phased sequences of intragenic regions, using SNPs. The key underlying idea of our method is to infer diploid sequences (a pair of phased sequences/alleles) at every functional locus utilizing the deep sequencing data from the 1000 Genomes Project and SNP data from the HapMap Project, and to build prediction models using flanking SNPs. Using this method, we have developed a database of prediction models for 611 known genes. Sequence prediction accuracy for these genes is 96.26% on average (ranges 79%-100%). This database of prediction models can be enhanced and scaled up to include new genes as the 1000 Genomes Project sequences additional genes on additional individuals. Applying our predictive model for the KCNJ11 gene to the Wellcome Trust Case Control Consortium (WTCCC) Type 2 diabetes cohort, we demonstrate how the prediction of phased sequences inferred from GWAS SNP genotype data can be used to facilitate interpretation and identify a probable functional mechanism such as protein changes.Prior to the general availability of routine sequencing of all subjects, the ISS method proposed here provides a time- and cost-effective approach to broadening the characterization of disease associated SNPs and regions, and facilitating the prioritization of candidate genes for more detailed functional and mechanistic studies.

Project description:Abstract Polycystic ovary syndrome (PCOS) is the most common cause of female infertility, affecting approximately 10 percent of women by Rotterdam criteria, and is comorbid with obesity, type II diabetes, hypertension, and non-alcoholic fatty liver disease. As twin studies reveal that genetics account for approximately 70% of PCOS risk, genome-wide association studies (GWAS) can provide powerful insight into PCOS etiology. PCOS GWAS studies from several populations identified a risk locus containing the FSHB gene, which encodes the beta subunit of follicle-stimulating hormone (FSH). As FSH supplementation can restore ovulation in some PCOS patients, deficient FSH signaling could be a causative factor of anovulation and potentially other facets of PCOS. Two of the lead single nucleotide polymorphisms (SNPs) in association with PCOS, rs11031005 and rs11031006, fall within a highly conserved genomic region in mammals. We hypothesized that the conserved region (~450 base pairs) enhances FSHB transcription, and that one or both PCOS-related SNPs alter its function. We have shown that the conserved region from both human and mouse can act as an enhancer of FSHB in LβT2 cells, an immortalized, mouse-derived, mature pituitary gonadotrope cell line, and that its function is altered by the rs11031006 minor allele through modification of an SF1 consensus site. As elimination of the SF1 site reduced but did not completely abolish the function of the enhancer, we continued our investigation to identify additional regulatory sites. Transient transfection of LβT2 cells revealed a possible role for the rs11031005 SNP in FSHB regulation, with the minor allele decreasing enhancer-mediated FSHB transcription. This effect may be due to decreased binding of an unidentified transcription factor, as gel shift revealed that the rs11031005 minor allele reduced the intensity of a binding complex. Using truncations and sliding deletions, we identified three additional putative transcription factor binding sites with consensus sequences for ZEB1, PTX1, and SMAD. To support a role for the conserved region as an enhancer in native chromatin, we assessed the histone status in LβT2 chromatin. Compared to the proximal Fshb promoter, the enhancer-specific marker, H3K4me1, was enriched near the conserved region. Neither promoter/enhancer markers of active (H3K27Ac) or repressed (H3K27me3) chromatin were enriched near the conserved region, although levels of both modifications were consistent with the Fshb proximal promoter. Overall, our data support the role of this conserved region as a novel regulator of FSHB/Fshb transcription and reveal a possible mechanism to explain the contribution of PCOS-associated SNPs through FSHB regulation.

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In-Silico Investigation of Effects of Single-Nucleotide Polymorphisms in PCOS-Associated CYP11A1 Gene on Mutated Proteins.

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