Vivekanandhan Subbiah

Homocystine Levels, Polymorphisms and the Risk of Ischemic Stroke in Young Asian Indians

BACKGROUND Homocysteine has been for a fairly long time been debated to be a risk factor for stroke. Opinions are divided as to whether raised levels of homocysteine seen in stroke patients are the cause or consequence of stroke. A large number of studies have been conducted in the Caucasian as well as on the Oriental population, which tend to suggest contradictory findings at many times. However, there have been no reports forthcoming from the Asian Indian population, which is a genetically different population than the previously studied populations. SUBJECTS AND METHODS In our present study, we looked at homocysteine levels and four commonly seen polymorphisms of homocysteine metabolizing enzymes and their respective prevalence in 120 acute onset ischemic stroke patients compared with an equal number of age and gender matched healthy population. We also tested the influence of folic acid dosage (5 mg OD) on the levels of homocysteine and the allied vitamin supplements, vitamin B12 and folate in smaller groups selected from the larger group. RESULTS AND CONCLUSIONS We found homocysteine levels to be significantly raised in the stroke population compared with healthy controls [patients: 12 micromol/L (range: 5.3-39.1 micromol/L), controls: 11.2 micromol/L (range: 6.2-14.2 micromol/L); P =0.001]. There was an almost total response to folic acid dosage as all hyperhomocysteinemic patients showed lowering of homocysteine levels in response to the dosage. The MTHFR 677 C > T polymorphisms showed association with both homocysteine levels as well as stroke (P < 0.001). Nutritional deficiency plays a dominant role in hyperhomocysteinemic conditions in our stroke population, however. Genetic determinants of homocysteine level may also have some part in determining hyperhomocysteinemic conditions in the Asian Indian populations.

Clinical effectiveness of rivastigmine monotherapy and combination therapy in Alzheimer's patients.

Rivastigmine is an acetylcholinesterase inhibitor; the genotype data seen alongside the phenotype data explain the mutation or the molecular genetics involved and also help to relate the phenotype of an individual with their genotype.

MRI-based polymer gel dosimetry for validating plans with multiple matrices in Gamma Knife stereotactic radiosurgery.

One of treatment planning techniques with Leksell GammaPlan (LGP) for Gamma Knife stereotactic radiosurgery (GKSRS) uses multiple matrices with multiple dose prescriptions. Computational complexity increases when shots are placed in multiple matrices with different grid sizes. Hence, the experimental validation of LGP calculated dose distributions is needed for those cases. For the current study, we used BANG3 polymer gel contained in a head‐sized glass bottle to simulate the entire treatment process of GKSRS. A treatment plan with three 18 mm shots and one 8 mm shot in separate matrices was created with LGP. The prescribed maximum dose was 8 Gy to three shots and 16 Gy to one of the 18 mm shots. The 3D dose distribution recorded in the gel dosimeter was read using a Siemens 3T MRI scanner. The scanning parameters of a CPMG pulse sequence with 32 equidistant echoes were as follows: TR=7s, echo step = 13.6 ms, field‐of‐view = 256 mm× 256 mm, and pixel size=1 mm×1 mm. Interleaved acquisition mode was used to obtain 15 to 45 2‐mm‐thick slices. Using a calibration relationship between absorbed dose and the spin‐spin relaxation rate (R2), we converted R2 images to dose images. MATLAB‐based in‐house programs were used for R2 estimation and dose comparison. Gamma‐index analysis for the 3D data showed gamma values less than unity for 86% of the voxels. Through this study we accomplished the first application of polymer gel dosimetry for a true comparison between measured 3D dose distributions and LGP calculations for plans using multiple matrices for multiple targets. PACS number: 87.53.Ly, 87.55‐x, 87.56 ‐g

Can Quantifying Free-Circulating DNA Be a Diagnostic and Prognostic Marker in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma?

PURPOSE Previous studies have reported significantly higher concentrations of serum DNA in various types of cancers. Thus the study aims to determine whether circulating free DNA (CFDNA) can aid in the diagnosis and prognosis of oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS A nonrandomized case-control study was planned where cases were derived from patients who presented to the KLE Institute of Dental Sciences, Belgaum, India, for evaluation and management of oral lesions between 2007 and 2009. In this study the predictor variable was status of the disease in the patients, and the outcome variable was CFDNA. Demographic variables included age, gender, tobacco consumption, and stage at diagnosis of cancer. Subjects with any known systemic disease, other tumors, or metastatic OSCC were excluded (CFDNA is altered in cases of tissue destruction and inflammatory diseases). The amount of CFDNA was determined through spectrophotometry (NanoDrop ND-1000 spectrophotometer; Thermo Fisher Scientific, Waltham, MA) in sampled blood and plasma. Mean and range for DNA quantification in plasma and blood were calculated in all groups and compared by use of the analysis of variance test. RESULTS A total of 390 cases (90 potentially malignant lesions, 150 OSCC cases, and 150 post-treatment OSCC cases) and 150 control subjects were studied. No significant difference was observed in levels of CFDNA in blood between the groups. Similarly, levels of CFDNA in plasma showed no significant difference except between the OSCC and potentially malignant groups, which was probably artifactual. CONCLUSIONS This study shows that disease progression in oral malignancy does not correlate with changes in levels of CFDNA in blood or plasma.

Association between GSTM1 and CYP1A1 polymorphisms and survival in oral cancer patients

AIMS Cancer patients inherited genotype may influence his or her survival, but evidence for the role of these genetic differences in oral cancer survival has not yet been explored. METHODS The authors evaluated polymorphisms in the GSTM1 and CYP1A1 genes for associations with overall survival in 100 oral squamous cell carcinoma (OSCC) treated patients and 100 controls who were followed up for survival within 2 years of the date of completion of their treatment. Overall survival was evaluated in Kaplan-Meier survival functions and Cox proportional hazards models. RESULTS After adjustment for stage and histology, GSTM1null genotype was associated with shorter survival among OSCC patients, compared with GSTM1 present genotype. There was no association between CYP1A1 C genotype and survival in the overall study population. CONCLUSION The study indicated a potential role for GSTM1 polymorphism in predicting the clinical outcomes of treated oral carcinoma patients.

Serological and Clinical Features of Patients with Myasthenia Gravis in North Indian Population

ABSTRACT Myasthenia gravis (MG) is a disorder of neuromuscular junction associated with presence of antibodies against nicotinic acetylcholine receptors (nAChRs). Here, we compared the clinical and serological profile of seropositive myasthenia gravis (SPMG) and seronegative myasthenia gravis (SNMG) patients. Anti-AChR antibody was measured using radio receptor immunoassay and correlated with clinical phenotype in 250 MG patients over 2004 and 2006. Out of 250 MG patients, 161 (64.4%%) were males (male:female == 1.8:1). SNMG patients formed 40%% (n == 101) of our MG patients which is much higher as compared to Caucasian and Oriental population (15%%–20%%). The median age of disease onset in SPMG was significantly higher than SNMG patients (43 years; range 8–74 vs. 35 years; range 4–72, p == .022). A bimodal peak of age of disease onset in both genders was observed (first peak in second–third decades and second one in fifth–sixth decades). Among the MG patients with late-onset of disease, male were significantly higher compared to Caucasian and Oriental MG population (p == .047). MG patients with thymoma were significantly older and consisted of higher percent of males. Bulbar symptoms and severe grade (IIB++ III++ IV) at disease onset were more frequent in SPMG than SNMG patients.

Elevated caspase 3 activity and cytosolic cytochrome c in NT2 cybrids containing amyotrophic lateral sclerosis subject mtDNA

Apoptosis of motor neurons is an important feature in amyotrophic lateral sclerosis (ALS). A vital role of mitochondria in apoptosis and cell survival is well documented. Eventually mitochondria have shown to be an early target in the pathogenesis of ALS. On account of these facts, we investigated the involvement of mitochondrial-dependent apoptosis in ALS and control (CTR) cybrids, generated fusing human platelets with mitochondrial DNA-depleted NT2-neuroteratocarcinoma cells. After a 6 week selection process during which transferred subject mtDNA repopulated the NT2 cells and restored mitochondrial oxygen consumption, we assessed cell viability and two programmed cell death parameters, caspase 3 activity and cytosolic cytochrome c levels. Compared to the control cybrid lines (n = 5), the ALS cybrid lines (n = 10) showed 45% less XTT reduction and higher caspase 3 activity ( p < 0.05, two-way Students t test) exhibiting lesser cell viability and execution of apoptosis. Elevated cytosolic cytochrome c levels in ALS cybrid lines (n = 8) than in CTR (n = 4) ( p < 0.05, two-way Students t-test) indicating its mitochondrial release and initiation of apoptosis. This indicates apoptosis as one of the possible mechanisms of cell death in ALS. Our findings support the view that in ALS, subjects mitochondria are altered in non-degenerating tissues in such a way that intrinsic apoptotic pathway activity is relatively increased.

Impact of CYP2D6 and CYP3A4 Genetic Polymorphism on Combined Cholinesterase Inhibitors and Memantine Treatment in Mild to Moderate Alzheimer's Disease

Aim: The impact of CYP2D6 and CYP3A4 polymorphism on the steady-state plasma concentrations and therapeutic outcome of donepezil monotherapy and combination therapy in Alzheimers disease (AD) patients. Methods: A total of 38 patients for donepezil and 17 patients for donepezil and memantine therapy, aged ≥55 years, were recruited meeting inclusion and exclusion criteria. Polymerase chain reaction-restriction fragment length polymorphism was performed. The liquid chromatography-tandem mass spectrometry method was used for estimation of drug levels of donepezil and memantine. Results: Significant allele frequency was observed for CYP2D6*3 polymorphism in patients on donepezil monotherapy and combination therapy. Significant allele frequency for CYP2D6*4 was observed in the patients on donepezil monotherapy. Conclusion: CYP2D6 polymorphism, though not significant, might partially be involved in the plasma concentration of AD drug.

Pharmacogenetics of uridine diphosphate glucuronosyltransferase (UGT2B7) genetic polymorphism on valproic acid pharmacokinetics in epilepsy

Background Sodium valproate is a widely prescribed broad-spectrum antiepileptic drug. It shows high inter-individual variability in pharmacokinetics and pharmacodynamics and has a narrow therapeutic range [1]. We evaluated the effects of polymorphic Uridine diphosphate glucuronosyltransferase (UGT2B7) metabolizing enzyme on the pharmacokinetics of sodium valproate in the patients with epilepsy who showed toxicity to therapy. Materials and methods Genotype analysis of the patients was made with polymerase chain–restriction fragment length polymorphism (RFLP) with sequencing. Plasma drug concentrations were measured with reversed phase high-performance liquid chromatography (HPLC) and concentration–time data were analyzed by using a non-compartmental approach. Results The results of this study suggested a significant genotypic as well as allelic association with valproic acid toxicity for UGT2B7 polymorphic enzymes. The elimination half-life (t1/2=42.2 h) of valproic acid was longer and the clearance rate (CL=947 ml/h) was lower in the poor metabolizers group of UGT2B7 polymorphism who showed toxicity than in the intermediate metabolizers group (t1/2 = 36.5 h, CL = 1,042 ml/h) or the extensive metabolizers group (t1/2 = 27. h, CL = 1,602 ml/h).Conclusions Our findings suggest that the UGT2B7 genetic polymorphism plays a significant role in the steady state concentration of valproic acid, and it thereby has an impact on the toxicity of the valproic acid used in the patients with epilepsy.

Silencing of Human CutC Gene (hCutC) Induces Apoptosis in HepG2 Cells.

Copper is an essential microelement required for maintaining normal cell physiology. Copper transporter CutC is one of the six members of Cut family proteins, involved in prokaryotic copper homeostasis. Human homolog of CutC (hCutC) is an intracellular copper-binding protein with unknown physiological function. In the present study using HepG2 cells, we report the effects of hCutC knockdown on copper sensitivity and morphology of cells that ultimately leads to apoptosis. We silenced hCutC using specific small interfering RNA (siRNA), and its downregulation was confirmed by quantitative real-time PCR. Though there was no significant variation in total cellular copper as estimated by inductively coupled plasma-atomic emission spectrometry (ICP-AES), knockdown of hCutC caused an increase in sensitivity of HepG2 cells to copper loads when compared to control cells (studied by MTT-based cell viability assay). Morphological analysis by transmission electron microscopy (TEM) indicated onset of apoptosis in hCutC-silenced cells which was exacerbated upon copper treatment. Mitochondrial transmembrane potential (ΔΨm) assay and DNA fragmentation assay further ensured apoptosis occurring in cells upon hCutC silencing. The present study reveals copper induced damage in cells upon hCutC silencing and provides evidence for the role of hCutC protein in intracellular copper homeostasis.