Impact of IL-17F 7488T/C Functional Polymorphism on Progressive Rheumatoid Arthritis: Novel Insight from the Molecular Dynamic Simulations.

Resorption of bones and cartilage coupled with structural changes in the inflamed joints are the major hallmark of rheumatoid arthritis (RA). Genetic polymorphisms in pro-inflammatory interleukins (ILs) appear to play an important role in the susceptibility towards progressive RA. We therefore aimed to investigate the association of single nucleotide polymorphisms (SNP), present in the hotspot coding/promoter regions of IL-6, -17 and -18, with RA susceptibility or severity in a larger study cohort from Pakistan together with finding clues as to how a functional SNP impacts the predisposition towards RA. TaqMan SNP genotyping approach was first used to assess IL-6 (rs1800795), IL-17 F (rs763780), IL-17A (rs2275913), and IL-18 (rs1946518) polymorphisms in 310 subjects (150 RA and 160 control). Molecular dynamic simulations (MDS) of wild- and mutant-type IL-17A with corresponding receptor were thereafter performed using AMBER-16; Chimera 1.13 was used for analyses. Our results showed the association of two SNPs, namely IL-6 - 174 G/C [allelic (OR = 0.960, 95% CI = 0.929-0.992, p = .009)] and IL-17 F 7488 T/C [allelic (OR = 0.907, 95%CI = 0.861-0.954, p = .000)] with increased RA risk in Pakistani subjects. When mapped, IL-17 F 7488 T/C was found involved in His161→Arg161 change near the C-terminus of IL-17 F. Comparative MDS revealed enhanced stability of the mutant hence advocating a potential role of IL-17F functional SNP in RA susceptibility and/or severity. This study provides a novel structural insight for SNP-derived functional mutation and its overall impact on binding with heterotrimeric receptor complex of IL-17 receptor thereby opening new avenues for understanding the biochemical basis of the disease.

Association of epilepsy and neurological impairments with homozygous recessive missense mutations found in the genes responsible for ganglioside biosynthesis (ST3GAL5) and calcium voltage-gated channels (CACNA1H) - insights through molecular dynamic simulations.

With over 2.2 million cases, the incidence rate of epilepsy in Pakistan is far higher than the rest of the world due primarily to the frequent, traditionally imposed cousin marriages. In the present study, comprehensive whole exome sequencing (WES) analyses of a three-generation family with four affected members presenting 'unexplained' childhood absence epilepsy (CAE), seizures and dementia, was performed in a quest to identify heritable, epilepsy-causal gene variants to better aid in carrier screening and genetic counselling. The WES data was generated, analyzed, and validated through Sanger's sequencing, molecular dynamic simulation (MDS) analysis, and molecular mechanics with generalized Born and surface area solvation (MM/GBSA) studies. Two homozygous recessive, missense mutations in ST3GAL5 (c.311A > G, p. His104Arg) and CACNA1H (c.6230G > A, p. Arg2077His) genes, earlier regarded as benign or of uncertain significance, have been identified as a potential etiology. Comparative MDS and free binding energy calculations revealed substantial structural perturbations in mutant forms of ST3GAL5 leading to decreased binding and reduced catalytic activity of the p.His104Arg and two other functional variants (p.Val74Glu and p.Arg288Ter) when compared with wild type. Our findings reinforce that WES analyses may uncover 'hidden', heritable variants and together with MDS and MM/GBSA may provide plausible clues to answer the unexplained causes of epilepsy for an effective management and better patient outcome. Further, revisit of epilepsy-associated mutational landscape in population context is imperative as the variants with 'benign' tags may turn out to be 'non-benign', when exist in combination with other benign.Communicated by Ramaswamy H. Sarma.

Molecular Dynamic Simulations Unravel the Underlying Impact of Missense Mutation in Autoimmunity Gene PTPN22 on Predisposition to Rheumatoid Arthritis.

Genetic mutations in various proteins have been implicated with increased risk or severity of rheumatoid arthritis (RA) in different population groups. In the present case-control study, we have investigated the risk association of single nucleotide mutations present in some of the highly reported anti-inflammatory proteins and/or cytokines, with RA susceptibility in the Pakistani subjects. The study involves 310 ethnically and demographically similar participants from whom blood samples were taken and processed for DNA extraction. Through extensive data mining, 5 hotspot mutations reported in 4 genes, that is, interleukin (IL)-4 (-590; rs2243250), IL-10 (-592; rs1800872), IL-10 (-1082; rs1800896), PTPN22 (C1858T; rs2476601), and TNFAIP3 (T380G; rs2230926), were selected for RA susceptibility analyses using genotyping assays. The results demonstrated the association of only 2 DNA variants [rs2243250 (odds ratio, OR = 2.025, 95% confidence interval, CI = 1.357-3.002, P = 0.0005 Allelic) and rs2476601 (OR = 4.25, 95% CI = 1.569-11.55, P = 0.004 Allelic)] with RA susceptibility in the local population. The former single nucleotide mutation was nonfunctional, whereas the latter, residing in the exonic region of a linkage-proven autoimmunity gene PTPN22, was involved in R620→W620 substitution. Comparative molecular dynamic simulations and free-energy calculations revealed a radical impact on the geometry/confirmation of key functional moieties in the mutant protein leading to a rather weak binding of W620 variant with the interacting receptor (SRC kinase). The interaction imbalance and binding instabilities provide convincing clues about the insufficient inhibition of T cell activation and/or ineffective clearance of autoimmune clones-a hallmark of several autoimmune disorders. In conclusion, the present research describes the association of 2 hotspot mutations in IL-4 promoter and PTPN22 gene with RA susceptibility in the Pakistani study cohort. It also details how a functional mutation in PTPN22 impacts the overall protein geometry, charge, and/or receptor interactions to contribute to RA susceptibility.

A novel missense compound heterozygous variant in TLR1 gene is associated with susceptibility to rheumatoid arthritis - structural perspective and functional annotations.

INTRODUCTION: Besides human leukocyte antigen (HLA-DRB1) locus, more than 100 loci across the genome have been identified and linked with the onset, expression and/or progression of rheumatoid arthritis (RA). However, there are still grey areas in our understanding of the key genetic contributors of the disease, particularly in familial cases. METHODS: In the present study, we have performed the whole exome sequencing (WES) of RA patients from two consanguineous families of Pakistan in a quest to identify novel, high-impact, RA-susceptibility genetic variants. RESULTS: Through stepwise filtering, around 17,000 variants (common in the affected members) were recognized, out of which 2651 were predicted to be deleterious. Of these, 196 had direct relevance to RA. When selected for homozygous recessive mode of inheritance, two novel pathogenic variants (c.1324T>C, p.Cys442→Arg442; c.2036T>C, p.Ile679→Thr679) in the TLR1 gene displayed the role of compound heterozygosity in modulating the phenotypic expression and penetrance of RA. The structural and functional consequences of the TLR1 missense single nucleotide mutations (Cys442→Arg442; Ile679→Thr679) were evaluated through molecular dynamic simulation (MDS) studies. Analysis showed domain's rigidification, conferring stability to mutant TLR1-TIR/TIRAP-TIR complex with concomitant increase in molecular interactions with pro-inflammatory cytokines, compared to the wild-type conformation. Gene co-expression network analysis highlighted interlinked partnering genes along with interleukin-6 production of TLR1 (corrected p-value 2.98e-4) and acetylcholine receptor activity of CHRNG (corrected p-value 6.12e-2) as highly enriched associated functions. CONCLUSION: The results, validated through case-control study subjects, suggested that the variants identified through WES and confirmed through Sanger sequencing and MDS are the novel disease variants and are likely to confer RA-susceptibility, independently and/or in a family-specific context. Key Points • Exploration of population based/ethno-specific big data is imperative to identify novel causal variants of RA. • Two new deleterious missense mutations in mutational hotspot exon 4 of TLR1 gene have been identified in Pakistani RA patients. • MD simulation data provides evidence for domain's rigidification, conferring stability to mutant TLR1-TIR/TIRAP-TIR complex, with concomitant increase in production of pro-inflammatory cytokines, thus adding to the onset/erosive outcome of RA.

Dysregulated Alanine as a Potential Predictive Marker of Glioma-An Insight from Untargeted HRMAS-NMR and Machine Learning Data.

Metabolic alterations play a crucial role in glioma development and progression and can be detected even before the appearance of the fatal phenotype. We have compared the circulating metabolic fingerprints of glioma patients versus healthy controls, for the first time, in a quest to identify a panel of small, dysregulated metabolites with potential to serve as a predictive and/or diagnostic marker in the clinical settings. High-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS-NMR) was used for untargeted metabolomics and data acquisition followed by a machine learning (ML) approach for the analyses of large metabolic datasets. Cross-validation of ML predicted NMR spectral features was done by statistical methods (Wilcoxon-test) using JMP-pro16 software. Alanine was identified as the most critical metabolite with potential to detect glioma with precision of 1.0, recall of 0.96, and F1 measure of 0.98. The top 10 metabolites identified for glioma detection included alanine, glutamine, valine, methionine, N-acetylaspartate (NAA), γ-aminobutyric acid (GABA), serine, α-glucose, lactate, and arginine. We achieved 100% accuracy for the detection of glioma using ML algorithms, extra tree classifier, and random forest, and 98% accuracy with logistic regression. Classification of glioma in low and high grades was done with 86% accuracy using logistic regression model, and with 83% and 79% accuracy using extra tree classifier and random forest, respectively. The predictive accuracy of our ML model is superior to any of the previously reported algorithms, used in tissue- or liquid biopsy-based metabolic studies. The identified top metabolites can be targeted to develop early diagnostic methods as well as to plan personalized treatment strategies.

Real-life effectiveness, safety, and tolerability of amlodipine/valsartan or amlodipine/valsartan/hydrochlorothiazide single-pill combination in patients with hypertension from Pakistan.

OBJECTIVE: EXCITE (clinical EXperienCe of amlodIpine and valsarTan in hypErtension) evaluated the real-life effectiveness, safety, and tolerability of single-pill combinations (SPCs) of amlodipine/valsartan (Aml/Val) and amlodipine/valsartan/hydrochlorothiazide (Aml/Val/HCTZ) in patients with hypertension from 13 countries in the Middle East and Asia. Here, we present data from Pakistan. METHODS: This was a 26-week observational, multicenter, prospective, open-label study. At week 26, change from baseline in mean sitting systolic (msSBP) and diastolic blood pressure (msDBP) and the proportion of patients achieving BP goal (SBP/DBP <140/90 mmHg; <130/80 mmHg in patients with diabetes) and response rates (SBP <140 mmHg [130 mmHg for patients with diabetes] or reduction of ≥20 mmHg; DBP <90 mmHg [80 mmHg for patients with diabetes] or reduction of ≥10 mmHg), were evaluated. Incidence of adverse events (AEs) and serious AEs (SAEs) was recorded as safety variables. Subjective assessment of effectiveness, compliance and tolerability was done by the physician. RESULTS: A total of 500 patients with hypertension (mean age of 48 years) were prescribed Aml/Val (n = 471, 94%) or Aml/Val/HCTZ (n = 29, 6%); 439 (87.8%) patients completed the study. At week 26, the mean BP decreased from 153.4/91.1 mmHg at baseline to 128.9/78.4 mmHg in the Aml/Val cohort (-24.5/-12.7 mmHg; p < 0.0001) and from 171.6/99.3 mmHg at baseline to 127.7/77.4 mmHg (-43.9/-21.9 mmHg; p < 0.0001) in the Aml/Val/HCTZ cohort. BP goals were achieved by 57% and 55.2% of patients in the Aml/Val and Aml/Val/HCTZ cohorts, respectively. A total of 40 (8%) patients reported at least one AE during the study period. Most common AEs included nausea (1.6%), headache (1.2%), vomiting (1.2%), and edema (1.2%). Most patients in Aml/Val cohort and all patients in Aml/Val/HCTZ cohort rated the effectiveness, compliance and tolerability as 'good' or 'very good'. CONCLUSIONS: Aml/Val with or without HCTZ in a SPC was effective and well-tolerated for BP reduction in this cohort of patients with hypertension from Pakistan.