Association of SUMOlation Pathway Genes With Stroke in a Genome-wide Association Study in India

OBJECTIVE: To undertake a genome-wide association study (GWAS) to identify genetic variants for stroke in an Indian population. METHODS: In a hospital-based case-control study, 8 teaching hospitals in India recruited 4,088 participants, including 1,609 stroke cases. Imputed genetic variants were tested for association with stroke subtypes using both single-marker and gene-based tests. Association with vascular risk factors was performed with logistic regression. Various databases were searched for replication, functional annotation, and association with related traits. Status of candidate genes previously reported in the Indian population was also checked. RESULTS: Associations of vascular risk factors with stroke were similar to previous reports and show modifiable risk factors such as hypertension, smoking, and alcohol consumption as having the highest effect. Single-marker–based association revealed 2 loci for cardioembolic stroke (1p21 and 16q24), 2 for small vessel disease stroke (3p26 and 16p13), and 4 for hemorrhagic stroke (3q24, 5q33, 6q13, and 19q13) at p < 5 × 10(−8). The index single nucleotide polymorphism of 1p21 is an expression quantitative trait locus (p(lowest) = 1.74 × 10(−58)) for RWDD3 involved in SUMOylation and is associated with platelet distribution width (1.15 × 10(−9)) and 18-carbon fatty acid metabolism (p = 7.36 × 10(−12)). In gene-based analysis, we identified 3 genes (SLC17A2, FAM73A, and OR52L1) at p < 2.7 × 10(−6). Eleven of 32 candidate gene loci studied in an Indian population replicated (p < 0.05), and 21 of 32 loci identified through previous GWAS replicated according to directionality of effect. CONCLUSIONS: This GWAS of stroke in an Indian population identified novel loci and replicated previously known loci. Genetic variants in the SUMOylation pathway, which has been implicated in brain ischemia, were identified for association with stroke

Association between methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and risk of ischemic stroke in North Indian population: A hospital based case–control study

Objective: The aim of the present case–control study was to determine the association between methylene tetrahydrofolate reductase (MTHFR) C677T (rs1801133) gene polymorphism and risk of ischemic stroke (IS) in North Indian population. Methods: Patients with IS and age-sex matched controls were recruited from Neurology Outpatient Department and Ward of All India Institute of Medical Sciences, New Delhi, India. Genotyping was performed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. PCR–RFLP results of nine randomly selected samples were confirmed by DNA sequencing. Genotypic and allelic distributions were compared between cases and controls. Statistical analysis was done by STATA, version 13.0 software. Results: Hypertension, diabetes, dyslipidemia, low socioeconomic status and family history of stroke were found to have an independent association with the risk of IS after adjusting for potential confounding factors. Mean age of cases and controls were 52.83 ± 12.59 and 50.97 ± 12.70 years. Multivariate logistic regression analysis showed an independent association between MTHFR C677T gene polymorphism and risk of IS (OR 1.91; 95% CI 1.07–3.41; p = 0.028) under dominant model [CT + TT vs. CC]. MTHFR C677T gene polymorphism was found to be independently associated with risk of small vessel disease (SVD) after adjustment for potential confounding factors [OR 2.51; 95% CI 1.30–4.85; p = 0.006] under the dominant model. Conclusion: Findings of the present study suggest that MTHFR C677T gene polymorphism might be a risk factor of IS mainly for SVD subtypes of IS in North Indian population. Further large prospective studies are required to confirm these findings

Neutralization of Tier-2 Viruses and Epitope Profiling of Plasma Antibodies from Human Immunodeficiency Virus Type 1 Infected Donors from India

<div><p>Broadly cross neutralizing antibodies (NAbs) are generated in a group of HIV-1 infected individuals during the natural infection, but little is known about their prevalence in patients infected with viral subtypes from different geographical regions. We tested here the neutralizing efficiency of plasma antibodies from 80 HIV-1 infected antiretroviral drug naive patients against a panel of subtype-B and C tier 2 viruses. We detected cross-neutralizing antibodies in approximately 19–27% of the plasma, however the subtype-C specific neutralization efficiency predominated (p = 0.004). The neutralizing activity was shown to be exclusively mediated by the immunoglobulin G (IgG) fraction in the representative plasma samples. Epitope mapping of three, the most cross-neutralizing plasma (CNP) AIIMS206, AIIMS239 and AIIMS249 with consensus-C overlapping envelope peptides revealed ten different binding specificities with only V3 and IDR being common. The V3 and IDR were highly antigenic regions but no correlation between their reciprocal Max50 binding titers and neutralization was observed. In addition, the neutralizing activity of CNP was not substantially reduced by V3 and gp41 peptides except a modest contribution of MPER peptide. The MPER was rarely recognized by plasma antibodies though antibody depletion and competition experiments demonstrated MPER dependent neutralization in two out of three CNP. Interestingly, the binding specificity of one of the CNP (AIIMS206) overlapped with broadly neutralizing mAb 2F5 epitope. Overall, the data suggest that, despite the low immunogenicity of HIV-1 MPER, the antibodies directed to this region may serve as crucial reagents for HIV-1 vaccine design.</p> </div

Depletion and competition of plasma antibodies from cross-neutralizing plasma.

<p>The depletion and competition of antibodies from three cross-neutralizing plasma (CNP) (AIIMS206, AIIMS239 and AIIMS249) was carried out with V3 (35 mer), MPER (25 mer) and IDR (19 mer) specific peptides. The antibodies from CNP were depleted by passing plasma samples (at 1∶30 dilution) over antigen coated ELISA plates (peptide coating concentration, 10 µg/ml) six times and the percent depletion of antibodies to each region was calculated as: percent depletion = 100−[100×(OD₄₀₅ at the last passage/OD₄₀₅ at the first passage)]. The percentage antibody depletion for CNP against each peptide is shown (A). As control, mock depletion of CNP was carried out on the uncoated plates which showed minimal effect on antibody depletion (A). For competition, CNP were preincubated 30 minutes with the same peptides at a final peptide concentration of 20 µg/ml. Both depleted (designated as ‘D’) and competed (designated as ‘+’) plasma were subsequently tested for neutralization with all eight viruses (data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043704#pone-0043704-t003" target="_blank">Table 3</a>). To validate the competition assay, we tested 447-52D an anti-V3 antibody known to neutralize SF162 (a tier 1 sensitive subtype-B virus) along with two peptides corresponding to consensus-C and B V3 sequences (at a final concentration, 10 µg/ml) (B).</p

Neutralization and binding of IgG fractions from cross-neutralizing (CNP) HIV-1 plasma.

<p>(A) Neutralization curves of IgG fractions purified from CNP on protein-A. The neutralization capacity of purified polyclonal IgG (open circle) is compared with those of the original plasma (solid circle). To allow the comparison, the purified IgG were concentrated to volumes equal to that of the original volume of plasma run over the column. Neutralization of CNP, AIIMS206, AIIMS239 and AIIMS249 column fractions is compared for the subtype C (Du156.12) and subtype B (JRFL) isolates. (B) The binding pattern of IgG fractions of CNP along with IgG from healthy control plasma A1. The ELISA binding was carried out with envelope recombinant gp120 proteins from subtype-A (92RW020), subtype-B (JRFL) and subtype-C (Du156.12) isolates. The anti-V3 (447-52D) and anti-B19 (1418) monoclonal antibodies were used as assay controls.</p

Epitope mapping of polyclonal antibodies from three cross-neutralizing plasma with consensus-C HIV-1 gp160 overlapping peptides.

<p>ELISA binding of polyclonal plasma antibodies from three most cross-neutralizing plasma (CNP) AIIMS206, AIIMS239 and AIIMS249 and two healthy seronegative individuals (A1 and A2) to 212 consensus-C HIV-1 envelope glycoprotein gp160 (gp120 and gp41) overlapping linear peptides (15 mer each with 11 amino acid overlap or 4 amino acid walk). The plasma were reacted at 4 dilutions (dilution range: 100 to 3000) and reciprocal Max50 binding titers were calculated using least square regression method. The plasma showed reactivity to second variable (V2), second constant (C2), third variable (V3), fourth constant-fifth variable (C4-V5), fifth constant (C5) regions of gp120 and fusion protein (FP), immunodominant region (IDR), C-heptad region (CHR), membrane proximal external region (MPER) and C-terminal (CT) of gp41 protein.</p