Anti-angiogenic effect of nano-formulated water soluble kaempferol and combretastatin in an in vivo chick chorioallantoic membrane model and HUVEC cells.

The present study evaluated the efficacy of nano-formulated water-soluble kaempferol and combretastatin alone and combined against the native kaempferol and combretastatin on angiogenesis. The solvent evaporation method was used to synthesize the nano-formulated water-soluble kaempferol and combretastatin and characterized using various analyses such as dynamic light scattering (DLS) and Fourier-transform infrared (FT-IR) spectroscopy.The anti-angiogenic activity of native, nano-formulated water-soluble kaempferol and combretastatin was investigated by cell viability on HUVEC and A498 cell lines, while chick chorioallantoic membrane (CAM) assay was utilized to assess morphometric and histopathological changes, and mRNA expressions of VEGF-A and FGF2 using qRT-PCR. MTT assay results revealed that the combination of nano-formulated water-soluble kaempferol and combretastatin significantly reduced the cell viability compared to control, individual treatments of native, nano-formulated water-soluble kaempferol, and combretastatin. Morphometric analysis of CAM showed that treatment with nano-formulated water-soluble kaempferol and combretastatin caused a substantial decrease in density, vessel network, branch points, and nets of CAM blood vessels. The histopathological results of CAM showed the irregular shape of blood vessels at the thin stratum of chronic endoderm, and blood capillaries were diminished compared to the control. In addition, the mRNA expression levels of VEGF-A and FGF2 were significantly decreased compared with native forms. Therefore, the findings of this study indicate that nano-formulated water-soluble combretastatin and kaempferol suppress angiogenesis by preventing the activation of endothelial cells and suppressing factors of angiogenesis. Moreover, a combination of nano-formulated water-soluble kaempferol and combretastatin worked much better than individual treatments.

Combining machine learning and structure-based approaches to develop oncogene PIM kinase inhibitors.

Introduction: PIM kinases are targets for therapeutic intervention since they are associated with a number of malignancies by boosting cell survival and proliferation. Over the past years, the rate of new PIM inhibitors discovery has increased significantly, however, new generation of potent molecules with the right pharmacologic profiles were in demand that can probably lead to the development of Pim kinase inhibitors that are effective against human cancer. Method: In the current study, a machine learning and structure based approaches were used to generate novel and effective chemical therapeutics for PIM-1 kinase. Four different machine learning methods, namely, support vector machine, random forest, k-nearest neighbour and XGBoost have been used for the development of models. Total, 54 Descriptors have been selected using the Boruta method. Results: SVM, Random Forest and XGBoost shows better performance as compared to k-NN. An ensemble approach was implemented and, finally, four potential molecules (CHEMBL303779, CHEMBL690270, MHC07198, and CHEMBL748285) were found to be effective for the modulation of PIM-1 activity. Molecular docking and molecular dynamic simulation corroborated the potentiality of the selected molecules. The molecular dynamics (MD) simulation study indicated the stability between protein and ligands. Discussion: Our findings suggest that the selected models are robust and can be potentially useful for facilitating the discovery against PIM kinase.

A comparative mRNA- and miRNA transcriptomics reveals novel molecular signatures associated with metastatic prostate cancers.

Background: Prostate cancer (PC) is a fatally aggressive urogenital cancer killing millions of men, globally. Thus, this study aims to identify key miRNAs, target genes, and drug targets associated with prostate cancer metastasis. Methods: The miRNA and mRNA expression datasets of 148 prostate tissue biopsies (39 tumours and 109 normal tissues), were analysed by differential gene expression analysis, protein interactome mapping, biological pathway analysis, miRNA-mRNA networking, drug target analysis, and survival curve analysis. Results: The dysregulated expression of 53 miRNAs and their 250 target genes involved in Hedgehog, ErbB, and cAMP signalling pathways connected to cell growth, migration, and proliferation of prostate cancer cells was detected. The subsequent miRNA-mRNA network and expression status analysis have helped us in narrowing down their number to 3 hub miRNAs (hsa-miR-455-3p, hsa-miR-548c-3p, and hsa-miR-582-5p) and 9 hub genes (NFIB, DICER1, GSK3B, DCAF7, FGFR1OP, ABHD2, NACC2, NR3C1, and FGF2). Further investigations with different systems biology methods have prioritized NR3C1, ABHD2, and GSK3B as potential genes involved in prostate cancer metastasis owing to their high mutation load and expression status. Interestingly, down regulation of NR3C1 seems to improve the prostate cancer patient survival rate beyond 150 months. The NR3C1, ABHD2, and GSK3B genes are predicted to be targeted by hsa-miR-582-5p, besides some antibodies, PROTACs and inhibitory molecules. Conclusion: This study identified key miRNAs (miR-548c-3p and miR-582-5p) and target genes (NR3C1, ABHD2, and GSK3B) as potential biomarkers for metastatic prostate cancers from large-scale gene expression data using systems biology approaches.

Structural characterization and conformational dynamics of alpha-1 antitrypsin pathogenic variants causing alpha-1-antitrypsin deficiency.

Background: Alpha-1 antitrypsin deficiency (A1ATD) is a progressive lung disease caused by inherited pathogenic variants in the SERPINA1 gene. However, their actual role in maintenance of structural and functional characteristics of the corresponding α-1 anti-trypsin (A1AT) protein is not well characterized. Methods: The A1ATD causative SERPINA1 missense variants were initially collected from variant databases, and they were filtered based on their pathogenicity potential. Then, the tertiary protein models were constructed and the impact of individual variants on secondary structure, stability, protein-protein interactions, and molecular dynamic (MD) features of the A1AT protein was studied using diverse computational methods. Results: We identified that A1ATD linked SERPINA1 missense variants like F76S, S77F, L278P, E288V, G216C, and H358R are highly deleterious as per the consensual prediction scores of SIFT, PolyPhen, FATHMM, M-CAP and REVEL computational methods. All these variants were predicted to alter free energy dynamics and destabilize the A1AT protein. These variants were seen to cause minor structural drifts at residue level (RMSD = <2Å) of the protein. Interestingly, S77F and L278P variants subtly alter the size of secondary structural elements like beta pleated sheets and loops. The residue level fluctuations at 100 ns simulation confirm the highly damaging structural consequences of all the six missense variants on the conformation dynamics of the A1AT protein. Moreover, these variants were also predicted to cause functional deformities by negatively impacting the binding energy of A1AT protein with NE ligand molecule. Conclusion: This study adds a new computational biology dimension to interpret the genotype-protein phenotype relationship between SERPINA1 pathogenic variants with its structural plasticity and functional behavior with NE ligand molecule contributing to the Alpha-1-antitrypsin deficiency. Our results support that A1ATD complications correlates with the conformational flexibility and its propensity of A1AT protein polymerization when misfolded.

Identification of miRNA-mRNA-TFs regulatory network and crucial pathways involved in asthma through advanced systems biology approaches.

Asthma is a life-threatening and chronic inflammatory lung disease that is posing a true global health challenge. The genetic basis of the disease is fairly well examined. However, the molecular crosstalk between microRNAs (miRNAs), target genes, and transcription factors (TFs) networks and their contribution to disease pathogenesis and progression is not well explored. Therefore, this study was aimed at dissecting the molecular network between mRNAs, miRNAs, and TFs using robust computational biology approaches. The transcriptomic data of bronchial epithelial cells of severe asthma patients and healthy controls was studied by different systems biology approaches like differentially expressed gene detection, functional enrichment, miRNA-target gene pairing, and mRNA-miRNA-TF molecular networking. We detected the differential expression of 1703 (673 up-and 1030 down-regulated) genes and 71 (41 up-and 30 down-regulated) miRNAs in the bronchial epithelial cells of asthma patients. The DEGs were found to be enriched in key pathways like IL-17 signaling (KEGG: 04657), Th1 and Th2 cell differentiation (KEGG: 04658), and the Th17 cell differentiation (KEGG: 04659) (p-values = 0.001). The results from miRNAs-target gene pairs-transcription factors (TFs) have detected the key roles of 3 miRs (miR-181a-2-3p; miR-203a-3p; miR-335-5p), 6 TFs (TFAM, FOXO1, GFI1, IRF2, SOX9, and HLF) and 32 miRNA target genes in eliciting autoimmune reactions in bronchial epithelial cells of the respiratory tract. Through systemic implementation of comprehensive system biology tools, this study has identified key miRNAs, TFs, and miRNA target gene pairs as potential tissue-based asthma biomarkers.

Granulomatous hepatitis in a Saudi child with IL2RA defect: a case report and literature review.

Interleukin-2 receptor alpha (IL2RA) defect (OMIM- # 606367) is an immune disease where affected patients are vulnerable to developing recurrent microbial infections in addition to lymphadenopathy and dermatological manifestations. This condition is known to be caused by pathogenic variants in the IL2RA gene, which are inherited in an autosomal recessive fashion. In this case report, we present a patient with IL2RA defect from Saudi Arabia who presented with chronic diarrhea, poor weight gain, mild villous atrophy, malnutrition, hepatomegaly, nonspecific inflammation, and an eczematous skin rash. His genetic analysis revealed a novel, homozygous, and likely pathogenic variant, that is, c.504 C>A (Cys168Ter), located in the exon 4of the IL2RA gene, which was inherited from his parents in an autosomal recessive mode of inheritance. This variant produces a 272-amino-acid shorter IL2RA protein chain, which most likely becomes degraded in the cytosol. Thus, we assume that the c.504 C>A is a null allele that abolishes the synthesis of IL2RA, malforms the IL-2 receptor complex, and eventually causes immunodeficiency manifestations. To our knowledge, this is the first time a person with IL2RA defect has shown signs of granulomatous hepatitis on a liver biopsy.

Computational approaches for discovering significant microRNAs, microRNA-mRNA regulatory pathways, and therapeutic protein targets in endometrial cancer.

Endometrial cancer (EC) is a urogenital cancer affecting millions of post-menopausal women, globally. This study aims to identify key miRNAs, target genes, and drug targets associated with EC metastasis. The global miRNA and mRNA expression datasets of endometrial tissue biopsies (24 tumors +3 healthy tissues for mRNA and 18 tumor +4 healthy tissues for miRNAs), were extensively analyzed by mapping of DEGs, DEMi, biological pathway enrichment, miRNA-mRNA networking, drug target identification, and survival curve output for differentially expressed genes. Our results reveal the dysregulated expression of 26 miRNAs and their 66 target genes involved in focal adhesions, p53 signaling pathway, ECM-receptor interaction, Hedgehog signaling pathway, fat digestion and absorption, glioma as well as retinol metabolism involved in cell growth, migration, and proliferation of endometrial cancer cells. The subsequent miRNA-mRNA network and expression status analysis have narrowed down to 2 hub miRNAs (hsa-mir-200a, hsa-mir-429) and 6 hub genes (PTCH1, FOSB, PDGFRA, CCND2, ABL1, ALDH1A1). Further investigations with different systems biology methods have prioritized ALDH1A1, ABL1 and CCND2 as potential genes involved in endometrial cancer metastasis owing to their high mutation load and expression status. Interestingly, overexpression of PTCH1, ABL1 and FOSB genes are reported to be associated with a low survival rate among cancer patients. The upregulated hsa-mir-200a-b is associated with the decreased expression of the PTCH1, CCND2, PDGFRA, FOSB and ABL1 genes in endometrial cancer tissue while hsa-mir-429 is correlated with the decreased expression of the ALDH1A1 gene, besides some antibodies, PROTACs and inhibitory molecules. In conclusion, this study identified key miRNAs (hsa-mir-200a, hsa-mir-429) and target genes ALDH1A1, ABL1 and CCND2 as potential biomarkers for metastatic endometrial cancers from large-scale gene expression data using systems biology approaches.

Molecular differential analysis of uterine leiomyomas and leiomyosarcomas through weighted gene network and pathway tracing approaches.

Uterine smooth muscular neoplastic growths like benign leiomyomas (UL) and metastatic leiomyosarcomas (ULMS) share similar clinical symptoms, radiological and histological appearances making their clinical distinction a difficult task. Therefore, the objective of this study is to identify key genes and pathways involved in transformation of UL to ULMS through molecular differential analysis. Global gene expression profiles of 25 ULMS, 25 UL, and 29 myometrium (Myo) tissues generated on Affymetrix U133A 2.0 human genome microarrays were analyzed by deploying robust statistical, molecular interaction network, and pathway enrichment methods. The comparison of expression signals across Myo vs UL, Myo vs ULMS, and UL vs ULMS groups identified 249, 1037, and 716 significantly expressed genes, respectively (p ≤ 0.05). The analysis of 249 DEGs from Myo vs UL confirms multistage dysregulation of various key pathways in extracellular matrix, collagen, cell contact inhibition, and cytokine receptors transform normal myometrial cells to benign leiomyomas (p value ≤ 0.01). The 716 DEGs between UL vs ULMS were found to affect cell cycle, cell division related Rho GTPases and PI3K signaling pathways triggering uncontrolled growth and metastasis of tumor cells (p value ≤ 0.01). Integration of gene networking data, with additional parameters like estimation of mutation burden of tumors and cancer driver gene identification, has led to the finding of 4 hubs (JUN, VCAN, TOP2A, and COL1A1) and 8 bottleneck genes (PIK3R1, MYH11, KDR, ESR1, WT1, CCND1, EZH2, and CDKN2A), which showed a clear distinction in their distribution pattern among leiomyomas and leiomyosarcomas. This study provides vital clues for molecular distinction of UL and ULMS which could further assist in identification of specific diagnostic markers and therapeutic targets.Abbreviations UL: Uterine Leiomyomas; ULMS: Uterine Leiomyosarcoma; Myo: Myometrium; DEGs: Differential Expressed Genes; RMA: Robust Multiarray Average; DC: Degree of Centrality; BC: Betweenness of Centrality; CGC: Cancer Gene Census; FDR: False Discovery Rate; TCGA: Cancer Genome Atlas; BP: Biological Process; CC: Cellular Components; MF: Molecular Function; PPI: Protein-Protein Interaction.

Molecular profiling of lamellar ichthyosis pathogenic missense mutations on the structural and stability aspects of TGM1 protein.

Lamellar ichthyosis (LI) is a rare inherited disease where affected infants present a extensive skin scaling characterized by hyperkeratosis. Inherited mutations in the Transglutaminase 1 (TGM1) protein is one of the known causative genetic factor for the LI. The main objective of this study is to explore the impact of LI causative missense mutations on the structural and stability aspects of TGM1 protein using structural modeling, molecular docking and molecular dynamics approaches. By testing all LI causative TMG1 mutations against multiple stability prediction methods, we found that L362R and L388P mutations positioned in the Transglut_core domain were most destabilizing to the stability of TGM1 protein. These 2 mutations were 3D protein modeled and further analyzed by molecular docking and dynamic simulation methods. Molecular docking of these TGM1 mutant structures with chitosan, a natural polyphenolic compound and known inducer for transglutaminase enzyme, has shown stable molecular interactions between the native TGM1-chitosan and TGM1(L388P)-chitosan complex, when compared to the TGM1(L362R)-chitosan complex. Interestingly, molecular dynamics analysis have also yielded similar findings, where L388P-chitosan complex is shown to develop B-sheets and attain better stability, whereas TGM1-L362R complex possessed coils over the simulation period, pointing its highly destabilizing behavior on the protein structure. This study concludes that missense mutations in Transglut_core domain of the TGM1 are deleterious to the stability and structural changes of TGM1 protein and also suggest that chitosan molecule could act as a natural activator against few pathogenic TGM1 mutations. Communicated by Ramaswamy H. Sarma.

Molecular design, synthesis and biological characterization of novel Resveratrol derivative as potential anticancer agent targeting NF-κB.

Resveratrol (RESV), an anticancer nutraceutical compound, is known to show poor bioavailability inside the human body. Therefore, this study has designed multiple chemical analogs of RESV compound for improving its pharmacokinetic as well as its anti-cancer properties. Initially, the drug likeliness and ADME-toxicity properties of these new chemical analogs were tested with the help of diverse computational approaches. Then the best predicted RESV derivative is synthesized by the organic method, and its NF-κB mediated anti-tumor activity assessed on histiocytic lymphoma U-937 cells. The new synthetic RESV analog, i.e. (E)-3-(prop-2-yn-1-yloxy)-5-(4-(prop-2-yn-1-yloxy) styryl) phenol has shown a rapid, persistent and better dose-dependent (IC50 of 7.25 µM) decrease in the viability of U937 cells than the native (IC50 of 30 µM) RESV compound. This analog has also demonstrated its potential ability in inducing apoptosis through DNA ladder formation. At 10 µg/ml concentration, this chemical derivative has shown a better NF-κB inhibition (IC50 is 2.45) compared to the native RESV compound (IC50 is 1.95). Molecular docking analysis found that this analog exerts its anti- NF-κB activity (binding energy of -6.78 kcal/mol and Ki 10 µM) by interacting with DNA binding residues (Arg246, Lys444, and Gln606) of p50 chain NF-κB. This study presents a novel RESV analog that could further develop as a potential anti-NF-κB mediated tumor inhibitor.

Structural prediction, whole exome sequencing and molecular dynamics simulation confirms p.G118D somatic mutation of PIK3CA as functionally important in breast cancer patients.

To understand the structural and functional importance of PIK3CA somatic mutations, whole exome sequencing, molecular dynamics simulation techniques in combination with in silico prediction algorithms such as SIFT, PolyPhen, Provean and CADD were employed. Twenty out of eighty missense somatic mutations in PIK3CA gene were found to be pathogenic by all the four algorithms. Most recurrent mutations found were known hotspot PIK3CA mutations with known clinical significance like p.E545 K, p.E545A, p.E545 G and p.C420R. A missense mutation p.G118D was found to be recurrently mutated in 5 cases. Interestingly, this mutation was observed in one of the patients who underwent whole exome sequencing and was completely absent from the controls. To see the effect of this mutation on the structure of PIK3CA protein, molecular dynamics simulation was performed. By molecular dynamics approach, we have shown that p.G118D mutation deviated from the native structure which was supported by the decrease in the number of hydrogen bonds, difference in hydrogen bond distance and angle, difference in root mean square deviation between the native and the mutant structures.

Molecular designing, virtual screening and docking study of novel curcumin analogue as mutation (S769L and K846R) selective inhibitor for EGFR.

The somatic mutations in ATP binding cleft of the tyrosine kinase binding domain of EGFR are known to occur in 15-40% of non-small cell lung cancer (NSCLC) patients. Although first and second generation anti-EGFR inhibitors are widely used to treat these patients, their therapeutic efficacy is modest and often results in adverse effects or drug resistance. Therefore, there is a need to develop novel as well as safe anti-EGFR drugs. The rapid emergence of computational drug designing provided a great opportunity to both discover and predict the efficacy of novel EGFR inhibitors from plant sources. In the present study, we designed several chemical analogues of edible curcumin (CUCM) compound and assessed their drug likeliness, ADME and toxicity properties using a diverse range of advanced computational methods. We also have examined the structural plasticity and binding characteristics of EGFR wild-type and mutant forms (S769L and K846R) against ligand molecules like Gefitinib, native CUCM, and different CUCM analogues. Through multidimensional experimental approaches, we conclude that CUCM-36 ((1E,4Z,6E)-1-(3,4-Diphenoxyphenyl)-5-hydroxy-7-(4-hydroxy-3-phenoxyphenyl)-1,4,6-heptatrien-3-one) is the best anti-EGFR compound with high drug-likeness, ADME properties, and low toxicity properties. CUCM-36 compound has demonstrated better affinity towards both wild-type (ΔG is -8.5 kcal/Mol) and mutant forms (V769L & K846R; ΔG for both is >-9.20 kcal/Mol) compared to natural CUCM and Gefitinib inhibitor. This study advises the future laboratory assays to develop CUCM-36 as a novel drug compound for treating EGFR positive non-small cell lung cancer patients.

Computational Molecular Phenotypic Analysis of PTPN22 (W620R), IL6R (D358A), and TYK2 (P1104A) Gene Mutations of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder of bone joints caused by the complex interplay between several factors like body physiology, the environment with genetic background. The recent meta-analysis of GWAS has expanded the total number of RA-associated loci to more than 100, out of which approximately ∼97% (98 variants) loci are located in non-coding regions, and the other ∼3% (3 variants) are in three different non-HLA genes, i.e., TYK2 (Prp1104Ala), IL6R (Asp358Ala), and PTPN22 (Trp620Arg). However, whether these variants prompt changes in the protein phenotype with regards to its stability, structure, and interaction with other molecules, remains unknown. Thus, we selected the three clinically pathogenic variants described above, as positive controls and applied diverse computational methods to scrutinize if those mutations cause changes in the protein phenotype. Both wild type and mutant protein structures of PTPN22 (W620R), IL6R (D358A), and TYK2 (P1104A) were modeled and studied for structural deviations. Furthermore, we have also studied the secondary structure characteristics, solvent accessibility and stability, and the molecular interaction deformities caused by the amino acid substitutions. We observed that simple nucleotide predictions of SIFT, PolyPhen, CADD and FATHMM yields mixed findings in screening the RA-missense variants which showed a ≥P-value threshold of 5 × 10-8 in genome wide association studies. However, structure-based analysis confirms that mutant structures shows subtle but significant changes at their core regions, but their functional domains seems to lose wild type like functional interaction. Our findings suggest that the multidirectional computational analysis of clinically potential RA-mutations could act as a primary screening step before undertaking functional biology assays.

Screening of common genetic variants in the APOB gene related to familial hypercholesterolemia in a Saudi population: A case-control study.

Familial hypercholesterolemia (FH) is a monogenic dominant inherited disorder of lipid metabolism characterized by elevated low-density lipoprotein levels, and is mainly attributable to mutations in low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proportein convertase subtilisin/kexin type 9 (PCSK9) genes. Next-generation and exome sequencing studies have primarily involved genome-wide association analyses, and meta-analyses and next-generation studies examined a few single-nucleotide polymorphisms (rs151009667 and Val2095Glu) in the ApoB gene. The present study was conducted to investigate the association of APOB and patients with FH in a Saudi population.We genotyped 100 patients with FH and 100 controls for 2 polymorphisms in APOB using polymerase chain reaction-restriction fragment length polymorphism, followed by 3% agarose gel electrophoresis. The strength of the association between the genotype and allele frequencies with the risk of developing FH was evaluated. Clinical details and genotype analysis results were recorded.For the rs151009667 polymorphism, 18% of the CT genotypes were observed only in patients with FH. There was a positive association between CT and CC (odds ratio [OR] 45.07 [95% conflict of interest (CI), 2.67-759.1]; P = .0001) and between T and C (OR 87.8 [95% CI, 5.34-144.2]; P < .0001). However, no Val2095Glu mutations were found in patients with FH or controls. There was also no correlation between clinical characteristics and the rs151009667 polymorphism.In conclusion, we confirmed the association between the rs151009667 polymorphism and FH in a Saudi population. The Val2095Glu novel variant did not appear in either patients with FH or controls. Similar studies should be performed in different ethnic populations to rule out the role of this polymorphism in FH.

Expanded Somatic Mutation Spectrum of MED12 Gene in Uterine Leiomyomas of Saudi Arabian Women.

MED12, a subunit of mediator complex genes is known to harbor genetic mutations, (mostly in exon 2), causal to the genesis of uterine leiomyomas among Caucasian, African American, and Asian women. However, the precise relationship between genetic mutations vs. protein or disease phenotype is not well-explained. Therefore, we sought to replicate the MED12 mutation frequency in leiomyomas of Saudi Arabian women, who represents ethnically and culturally distinct population. We performed molecular screening of MED12 gene (in 308 chromosomes belonging to 154 uterine biopsies), analyzed the genotype-disease phenotype correlations and determined the biophysical characteristics of mutated protein through diverse computational approaches. We discovered that >44% (34/77) leiomyomas of Arab women carry a spectrum of MED12 mutations (30 missense, 1 splice site, and 3 indels). In addition to known codon 44, we observed novel somatic mutations in codons 36, 38, and 55. Most genetically mutated tumors (27/30; 90%) demonstrated only one type of genetic change, highlighting that even single allele change in MED12 can have profound impact in transforming the normal uterine myometrium to leiomyomas. An interesting inverse correlation between tumor size and LH is observed when tumor is positive to MED12 mutation (p < 0.05). Our computational investigations suggest that amino acid substitution mutations in exon-2 region of MED12 might contribute to potential alterations in phenotype as well as the stability of MED12 protein. Our study, being the first one from Arab world, confirms the previous findings that somatic MED12 mutations are critical to development and progression of uterine leiomyomas irrespective of the ethnic background. We recommend that mutation screening, particularly codon 44 of MED12 can assist in molecular diagnostics of uterine leiomyomas in majority of the patients.

Distribution of CYP2C8 and CYP2C9 amino acid substitution alleles in South Indian diabetes patients: A genotypic and computational protein phenotype study.

The CYP2C8 and CYP2C9 are two major isoforms of the cytochrome P450 enzyme family, which is involved in drug response, detoxification, and disease development. This study describes the differential distribution of amino acid substitution variants of CYP2C8 (*2-I269F & *3-R139K) and CYP2C9 (*2-C144R & *3-L359A) genes in 234 type 2 diabetes mellitus (T2DM) patients and 218 healthy controls from Andhra Pradesh, South India. Single locus genotype analysis has revealed that homozygous recessive genotypes of 2C8*2-TT (P ≤ .03), 2C9*2-TT (P ≤ .02), and heterozygous 2C9*3-AC (P ≤ .006) are seen to be increasingly present in the case group, indicating a significant level of their association with diabetes in Andhra population. The statistical significance of these recessive genotypes has persisted even under their corresponding allelic forms (P ≤ .01). Genotype association results were further examined by computational protein structure and stability analysis to assess the deleteriousness of the amino acid changes. The mutant CYP 2C8 and 2C9 (both *2 and *3) proteins showed structural drifts at both amino acid residue (range 0.43Å-0.77Å), and polypeptide chain levels (range 0.68Å-1.81Å) compared to their wild-type counterparts. Furthermore, the free energy value differences (range -0.915 to -1.38 Kcal/mol) between mutant and native protein structures suggests the deleterious and destabilizing potential of amino acid substitution polymorphisms of CYP genes. The present study confirms the variable distribution of CYP2C8 (*2 and *3) and CYP2C9 (*2 and *3) allelic polymorphisms among South Indian diabetic populations and further warrants the serious attention of CYP gene family, as a putative locus for disease risk assessment and therapy.

Whole exome sequencing of a consanguineous family identifies the possible modifying effect of a globally rare AK5 allelic variant in celiac disease development among Saudi patients.

Celiac disease (CD), a multi-factorial auto-inflammatory disease of the small intestine, is known to occur in both sporadic and familial forms. Together HLA and Non-HLA genes can explain up to 50% of CD's heritability. In order to discover the missing heritability due to rare variants, we have exome sequenced a consanguineous Saudi family presenting CD in an autosomal recessive (AR) pattern. We have identified a rare homozygous insertion c.1683_1684insATT, in the conserved coding region of AK5 gene that showed classical AR model segregation in this family. Sequence validation of 200 chromosomes each of sporadic CD cases and controls, revealed that this extremely rare (EXac MAF 0.000008) mutation is highly penetrant among general Saudi populations (MAF is 0.62). Genotype and allelic distribution analysis have indicated that this AK5 (c.1683_1684insATT) mutation is negatively selected among patient groups and positively selected in the control group, in whom it may modify the risk against CD development [p<0.002]. Our observation gains additional support from computational analysis which predicted that Iso561 insertion shifts the existing H-bonds between 400th and 556th amino acid residues lying near the functional domain of adenylate kinase. This shuffling of amino acids and their H-bond interactions is likely to disturb the secondary structure orientation of the polypeptide and induces the gain-of-function in nucleoside phosphate kinase activity of AK5, which may eventually down-regulates the reactivity potential of CD4+ T-cells against gluten antigens. Our study underlines the need to have population-specific genome databases to avoid false leads and to identify true candidate causal genes for the familial form of celiac disease.

Replication of GWAS loci revealed the moderate effect of TNRC6B locus on susceptibility of Saudi women to develop uterine leiomyomas.

AIM: Uterine leiomyomas (UL) are smooth muscular nodes, whose growth is dependant up on the complex interplay of hormones with genes and uterine physiology. Global statistics indicate the role of ethnic and racial background as contributory factors for UL development. Owing to the lack of data, this study aimed to examine the association between genetic polymorphisms and susceptibility of Arab women of developing UL. METHODS: We genotyped 105 UL patients and 112 healthy controls for five genetic polymorphisms through real time PCR method. The strength of the association between genotype and allele frequencies with risk of developing UL was tested with their χ2 and odds ratio (OR) values. The synergistic cooperation between genetic polymorphisms was estimated through multifactor dimensionality reduction assay. RESULTS: We found that Saudi women with the AG genotype for the rs12484776 polymorphism are at a 2.6-fold higher risk of developing UL compared to those with other genotypes (OR, 2.69; 95% confidence interval [CI]: 1.45-5.00; P < 0.001). This significance persisted even under co-dominant models (i.e., AA vs GG + AG [OR, 2.74; 95%CI: 1.48-5.08; P = 0.001; and AG vs GG + AG [OR, 2.41; 95% CI: 1.33-4.39; P = 0.003). Genotype distribution frequencies for rs1056836, rs7913069, rs2280543, and rs4247357 were not shown to elevate the disease risk (for all tests P > 0.05). CONCLUSION: The rs12484776 significantly contributes to UL risk among Saudi women, both in single and also in synergistic cooperation with rs2280543, rs7913069, and rs1056836 markers. Our results have yielded mixed findings in replicating European- and Japanese-specific UL genetic susceptibility loci among a geographically and culturally distinct population of the Saudi Arabian Peninsula.

A Computational Protein Phenotype Prediction Approach to Analyze the Deleterious Mutations of Human MED12 Gene.

Genetic mutations in MED12, a subunit of Mediator complex are seen in a broad spectrum of human diseases. However, the underlying basis of how these pathogenic mutations elicit protein phenotype changes in terms of 3D structure, stability and protein binding sites remains unknown. Therefore, we aimed to investigate the structural and functional impacts of MED12 mutations, using computational methods as an alternate to traditional in vivo and in vitro approaches. The MED12 gene mutations details and their corresponding clinical associations were collected from different databases and by text-mining. Initially, diverse computational approaches were applied to categorize the different classes of mutations based on their deleterious impact to MED12. Then, protein structures for wild and mutant types built by integrative modeling were analyzed for structural divergence, solvent accessibility, stability, and functional interaction deformities. Finally, this study was able to identify that genetic mutations mapped to exon-2 region, highly conserved LCEWAV and Catenin domains induce biochemically severe amino acid changes which alters the protein phenotype as well as the stability of MED12-CYCC interactions. To better understand the deleterious nature of FS-IDs and Indels, this study asserts the utility of computational screening based on their propensity towards non-sense mediated decay. Current study findings may help to narrow down the number of MED12 mutations to be screened for mediator complex dysfunction associated genetic diseases. This study supports computational methods as a primary filter to verify the plausible impact of pathogenic mutations based on the perspective of evolution, expression and phenotype of proteins. J. Cell. Biochem. 117: 2023-2035, 2016. © 2016 Wiley Periodicals, Inc.

Correlation between EGFR Gene Mutations and Lung Cancer: a Hospital-Based Study.

Epidermal growth factor receptor (EGFR) is one of the targeted molecular markers in many cancers including lung malignancies. Gefitinib and erlotinib are two available therapeutics that act as specific inhibitors of tyrosine kinase (TK) domains. We performed a case-control study with formalin-fixed paraffin-embedded tissue blocks (FFPE) from tissue biopsies of 167 non-small cell lung carcinoma (NSCLC) patients and 167 healthy controls. The tissue biopsies were studied for mutations in exons 18-21 of the EGFR gene. This study was performed using PCR followed by DNA sequencing. We identified 63 mutations in 33 men and 30 women. Mutations were detected in exon 19 (delE746-A750, delE746-T751, delL747-E749, delL747-P753, delL747-T751) in 32 patients, exon 20 (S786I, T790M) in 16, and exon 21 (L858R) in 15. No mutations were observed in exon 18. The 63 patients with EFGR mutations were considered for upfront therapy with oral tyrosine kinase inhibitor (TKI) drugs and have responded well to therapy over the last 15 months. The control patients had no mutations in any of the exons studied. The advent of EGFR TKI therapy has provided a powerful new treatment modality for patients diagnosed with NSCLC. The study emphasizes the frequency of EGFR mutations in NSCLC patients and its role as an important predictive marker for response to oral TKI in the south Indian population.