Subhadip Chakraborty

Association of IL1A and IL1B loci with primary open angle glaucoma

BackgroundRecent studies suggest that glaucoma is a neurodegenerative disease in which secondary degenerative losses occur after primary insult by raised Intraocular pressure (IOP) or by other associated factors. It has been reported that polymorphisms in the IL1A and IL1B genes are associated with Primary Open Angle Glaucoma (POAG). The purpose of our study was to investigate the role of these polymorphisms in eastern Indian POAG patients.MethodsThe study involved 315 unrelated POAG patients, consisting of 116 High Tension Glaucoma (HTG) patients with intra ocular pressure (IOP) > 21 mmHg and 199 non-HTG patients (presenting IOP < 20 mmHg), and 301 healthy controls from eastern India. Genotypes were determined by polymerase chain reaction and restriction digestion for three single nucleotide polymorphisms (SNPs): IL1A (-889C/T; rs1800587), IL1B (-511C/T; rs16944) and IL1B (3953C/T; rs1143634). Haplotype frequency was determined by Haploview 4.1 software. The association of individual SNPs and major haplotypes was evaluated using chi-square statistics. The p-value was corrected for multiple tests by Bonferroni method.ResultsNo significant difference was observed in the allele and genotype frequencies for IL1A and IL1B SNPs between total pool of POAG patients and controls. However, on segregating the patient pool to HTG and non-HTG groups, weak association was observed for IL1A polymorphism (-889C/T) where -889C allele was found to portray risk (OR = 1.380; 95% CI = 1.041-1.830; p = 0.025) for non-HTG patients. Similarly, 3953T allele of IL1B polymorphism (+3953C/T) was observed to confer risk to HTG group (OR = 1.561; 95% CI = 1.022-2.385; p = 0.039). On haplotype analysis it was observed that TTC was significantly underrepresented in non-HTG patients (OR = 0.538; 95% CI = 0.356- 0.815; p = 0.003) while TCT haplotype was overrepresented in HTG patients (OR = 1.784; 95% CI = 1.084- 2.937; p = 0.022) compared to control pool. However, after correction for multiple tests by Bonferroni method, an association of only TTC haplotype with non-HTG cases sustained (pcorrected = 0.015) and expected to confer protection.ConclusionThe study suggests that the genomic region containing the IL1 gene cluster influences the POAG pathogenesis mostly in non-HTG patients in eastern India. A similar study in additional and larger cohorts of patients in other population groups is necessary to further substantiate the observation.

Evaluation of genetic association of the INK4 locus with primary open angle glaucoma in East Indian population.

INK4 locus at chromosome 9p21 has been reported to be associated with primary open angle glaucoma (POAG) and its subtypes along with the associated optic disc parameters across the populations of European, Japanese and African ancestries. The locus encodes three tumor suppressor genes namely CDKN2A, ARF, CDKN2B and a long non-coding RNA CDKN2B-AS1 (also known as ANRIL). Here, we report association study of 34 SNPs from INK4 locus with POAG in a population of Indo-European ancestry from the eastern part of India (350 patients and 354 controls). With 81% power to detect genetic association we observed only nominal association of rs1011970 (uncorrected p = 0.048) with POAG and rs10120688 (uncorrected p = 0.048) in patients without a high intra-ocular pressure (IOP<21 mm of Hg) compared to controls. This study, in contrast to the previous reports, suggests lack of significant genetic association of INK4 locus with POAG in East Indian population which needs to be replicated in larger studies in diverse world populations.

Gene-rich large deletions are overrepresented in POAG patients of Indian and Caucasian origins.

PURPOSE Large copy number variations (CNV) can contribute to increased burden for neurodegenerative diseases. In this study, we analyzed the genome-wide burden of large CNVs > 100 kb in primary open angle glaucoma (POAG), a neurodegenerative disease of the eye that is the largest cause of irreversible blindness. METHODS Genome-wide analysis of CNVs > 100 kb were analyzed in a total of 1720 individuals, including an Indian cohort (347 POAG cases and 345 controls) and a Caucasian cohort (624 cases and 404 controls). All the CNV data were obtained from experiments performed on Illumina 660W-Quad (infinium) arrays. RESULTS We observed that for both the populations CNVs > 1 Mb was significantly enriched for gene-rich regions unique to the POAG cases (P < 10(-11)). In the Indian cohort CNVs > 1 Mb (39 calls) in patients influenced 125 genes while in controls 31 such CNVs influenced only 5 genes with no overlap. In both cohorts we observed 1.9-fold gene enrichment in patients for deletions compared to duplications, while such a bias was not observed in controls (0.3-fold). Overall duplications > 1 Mb were more than deletions (Del/Dup = 0.82) confirming that the enrichment of gene-rich deletions in patients was associated with the disease. Of the 39 CNVs > 1 Mb from Indian patients, 28 (72%) also were implicated in other neurodegenerative disorders, like autism, schizophrenia, sensorineural hearing loss, and so forth. We found one large duplication encompassing CNTN4 gene in Indian and Caucasian POAG patients that was absent in the controls. CONCLUSIONS To our knowledge, our study is the first report on large CNV bias for gene-rich regions in glaucomatous neurodegeneration, implicating its impact across populations of contrasting ethnicities. We identified CNTN4 as a novel candidate gene for POAG.

Investigating the quasi-oscillatory behavior of electrical parameters with the concentration of D-glucose in aqueous solution

Abstract The impedance, capacitance and conductance of deionized water-glucose polar solution is measured by employing impedance spectroscopy and a quasi-oscillatory nature of variation with glucose content in the solution is observed. Such quasi-oscillatory nature is attributed to the randomly distributed water-water, water-glucose and glucose-glucose dipole interactions at the molecular level in the solution. A relevant analytical model is developed on the basis of such random distribution of the molecular dipoles and the experimental data agree well with those obtained from the theoretical model. The electrical parameters are measured in the frequency range of 100Hz to 4MHz for the volume fractions of glucose with respect to water in the range of 0.1 to 0.5. The impedance, capacitance and conductance are obtained to be in the range of 1.03 kΩ – 112 kΩ, 34.9 pF – 1.66 nF, and 8.95 μS – 52.9 μS respectively for the glucose volume fraction range considered.

Functional and Structural Analyses of CYP1B1 Variants Linked to Congenital and Adult-Onset Glaucoma to Investigate the Molecular Basis of These Diseases

Glaucoma, the leading cause of irreversible blindness, appears in various forms. Mutations in CYP1B1 result in primary congenital glaucoma (PCG) by an autosomal recessive mode of inheritance while it acts as a modifier locus for primary open angle glaucoma (POAG). We investigated the molecular basis of the variable phenotypes resulting from the defects in CYP1B1 by using subclones of 23 CYP1B1 mutants reported in glaucoma patients, in a cell based system by measuring the dual activity of the enzyme to metabolize both retinol and 17β-estradiol. Most variants linked to POAG showed low steroid metabolism while null or very high retinol metabolism was observed in variants identified in PCG. We examined the translational turnover rates of mutant proteins after the addition of cycloheximide and observed that the levels of enzyme activity mostly corroborated the translational turnover rate. We performed extensive normal mode analysis and molecular-dynamics-simulations-based structural analyses and observed significant variation of fluctuation in certain segmental parts of the mutant proteins, especially at the B-C and F-G loops, which were previously shown to affect the dynamic behavior and ligand entry/exit properties of the cytochrome P450 family of proteins. Our molecular study corroborates the structural analysis, and suggests that the pathologic state of the carrier of CYP1B1 mutations is determined by the allelic state of the gene. To our knowledge, this is the first attempt to dissect biological activities of CYP1B1 for correlation with congenital and adult onset glaucomas.

Analysis of COCH and TNFA Variants in East Indian Primary Open-Angle Glaucoma Patients

Glaucoma represents a heterogeneous group of optic neuropathies with a complex genetic basis. It is the second-largest cause of blindness in the world that reduces vision without warning and often without symptoms. Among 3 major subtypes of glaucoma, primary open-angle glaucoma (POAG) is the most common form. The focus of this study is to understand the molecular basis of the disease among Indian patients with respect to two genes, Cochlin (COCH) and tumor necrosis factor alpha (TNFA), selected based on reports of possible association with POAG. The genes were screened in patients and controls by PCR and direct sequencing. Although two novel changes (–450 C/T and –79 G/G) were identified in the 5′upstream region of COCH, no causal variant could be identified in either gene. –450 C/T was detected in 3 patients and 2 controls and –79 G/C in a single patient. Further, we did not observe significant association with the promoter SNPs of TNFA that had been previously reported to be associated with POAG pathogenesis. Thus, our study suggests lack of association of both COCH and TNFA with POAG pathogenesis.

FT-MIR supported Electrical Impedance Spectroscopy based study of sugar adulterated honeys from different floral origin

This study sought to detect the presence of sucrose as an adulterant in selected honey varieties from different floral origins by employing Electrical Impedance Spectroscopy (EIS) technique which has been simultaneously supported by Fourier Transform-Mid Infrared Spectroscopy (FT-MIR) measurements to provide a rapid, robust yet simple platform for honey quality evaluation. Variation of electrical parameters such as impedance, capacitance and conductance for 10%, 20%, 30%, 40%, 50%, 60% and 70% (w/w) sucrose syrup (SS) adulterated honey samples are analyzed and their respective current-voltage (I-V) characteristics are studied. Capacitance, conductance and net current flowing through the system are observed to decrease linearly whereas system impedance has been found to increase similarly with the increase in adulterant content. Also, FT-MIR measurements in the spectral region between 1800cm-1 and 650cm-1 reveal the increment of absorbance values due to the addition of SS. Full-Width-at-Half-Maximum (FWHM) is estimated from the spectral peak 1056cm-1 for all pure and adulterated honey samples and is observed to be linearly increasing with increase in adulterant content. Finally, the coefficient of sensitivity has been extracted for all varieties of honey considered in terms of the measured conductance values.

Dielectric properties of plasma membrane: A signature for dyslipidemia in diabetes mellitus

Dielectric properties of a living biological membrane play crucial role indicating the status of the cell in pathogenic or healthy condition. A distinct variation in membrane capacitance and impedance was observed for peripheral blood mononuclear cell (PBMC) suspensions for diabetic and diabetic-dyslipidemic subjects compared to healthy control. Low frequency region were explicitly considered in electrical analysis to address complex membrane dielectric factors that alter the system capacitance of a PBMC suspension. Such variation was marked in size, morphology and membrane function of PBMCs for control and diseased cases. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) studies reveal significant alteration in surface morphology of PBMCs in diseased condition. Side scatter of flow cytometry reveals complexity of PBMCs in diseased condition. Changes in size between groups were not found by SEM and forward scatter. Functional alteration in PBMCs was manifested by significant changes in cell membrane properties like Na+, K+ ATPase and Ca2+, Mg2+ ATPase activity, reduced plasma membrane fluidity and changes in intracellular Ca2+ content, which bear significant correlation in diabetic and diabetic dyslipidemic subjects. Therefore, dielectric parameters of PBMCs in diabetic-dyslipidemic challenges may led to interesting correlation opening the possibility of identifying crucial signature biomarkers.