Association of DNA repair gene XPC Ala499Val (rs2228000 C>T) and Lys939Gln (rs2228001 A>C) polymorphisms with the risk of chronic myeloid leukemia: A case-control study in a South Indian population.

BACKGROUND: Xeroderma pigmentosum complementation group C (XPC), a DNA repair protein, plays an important role in the maintenance of genomic integrity and is essential for the nucleotide excision repair pathway. Polymorphisms in the XPC gene may alter DNA repair leading to genetic instability and oncogenesis. The present study aimed to assess the relationship between the XPC Ala499Val (rs2228000 C>T) and Lys939Gln (rs2228001 A>C) non-synonymous polymorphisms and susceptibility to chronic myeloid leukemia (CML) pathogenesis, disease progression and the response to targeted therapeutic regimen, imatinib mesylate. METHODS: This case-control study included 212 cases and 212 controls, and the genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism assays. RESULTS: Our results showed significant association of variant CT (odds ratio = 1.92, 95% confidence interval = 1.21-3.06, p = 0.003) and TT (odds ratio = 2.84, 95% confidence interval = 1.22-6.71, p = 0.007) genotypes in patients with the XPC Ala499Val polymorphism and CML risk. In addition, these genotypes were associated with CML progression to advanced phases (p = 0.006), splenomegaly (p = 0.017) and abnormal lactate dehydrogenase levels (p = 0.03). XPC Lys939Gln was found to correlate with a poor response to therapy, showing borderline significant association with minor cytogenetic response (p = 0.08) and a poor molecular response (p = 0.06). Significant association of the Ala499Val and Lys939Gln polymorphisms with prognosis was observed (Hasford high risk, p = 0.031 and p = 0.019, respectively). Haplotype analysis showed a strong correlation of variant TC haplotype with poor therapy responses (minor cytogenetic response, p = 0.019; poor molecular response, p < 0.0001). CONCLUSIONS: In conclusion, our results suggest that XPC Ala499Val is a high-penetrance CML susceptibility polymorphism. Both polymorphisms studied are considered as genetic markers with respect to assessing disease progression, therapy response and prognosis in CML patients.

Molecular insights into the coding region mutations of low-density lipoprotein receptor adaptor protein 1 (LDLRAP1) linked to familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is a lipid disorder caused by pathogenic mutations in LDLRAP1 gene. The present study has aimed to deepen our understanding about the pathogenicity predictions of FH causative genetic mutations, as well as their relationship to phenotype changes in LDLRAP1 protein, by utilizing multidirectional computational analysis. METHODS: FH linked LDLRAP1 mutations were mined from databases, and the prediction ability of several pathogenicity classifiers against these clinical variants, was assessed through different statistical measures. Furthermore, these mutations were 3D modelled in protein structures to assess their impact on protein phenotype changes. RESULTS: Our findings suggest that Polyphen-2, when compared with SIFT, M-CAP and CADD tools, can make better pathogenicity predictions for FH causative LDLRAP1 mutations. Through, 3D simulation and superimposition analysis of LDLRAP1 protein structures, it was found that missense mutations do not create any gross changes in the protein structure, although they could induce subtle structural changes at the level of amino acid residues. Near native molecular dynamic analysis revealed that missense mutations could induce variable degrees of fluctuation differences guiding the protein flexibility. Stability analysis showed that most missense mutations shifts the free energy equilibrium and hence they destabilize the protein. Molecular docking analysis demonstrates the molecular shifts in hydrogen and ionic bonds and Van der waals bonding properties, which further cause differences in the binding energy of LDLR-LDLRAP1 proteins. CONCLUSIONS: The diverse computational approaches used in the present study may provide a new dimension for exploring the structure-function relationship of the novel and deleterious LDLRAP1 mutations linked to FH.

PTEN and p16 genes as epigenetic biomarkers in oral squamous cell carcinoma (OSCC): a study on south Indian population.

Phosphatase and tensin homolog (PTEN) and p16INK4a (p16) genes are tumor suppressor genes, associated with epigenetic alterations. PTEN and p16 promoter hypermethylation is a major epigenetic silencing mechanism leading to cancer. The cooperation between PTEN and p16 in pathogenesis of cancers suggest that their combination might be considered as potential molecular marker for specific subgroups of patients. Hence, the present study aimed to investigate whether PTEN and p16 promoter methylations were involved in oral squamous cell carcinoma (OSCC) in south Indian subjects. DNA methylation quantitative analyses of the two candidate tumor suppressor genes PTEN and p16 were performed by methylation-specific polymerase chain reaction (MSP). Fifty OSCC biopsy samples and their corresponding non-malignant portions as controls were studied comparatively. The methylation status was correlated with the clinical manifestations. Twelve out of 50 patients (24 %) were found to be methylated for PTEN gene, whereas methylation of the p16 gene occurred in 19 out of 50 cases (38 %). A statistically significant result was obtained (P = <0.0001 and 0.017) for both PTEN and p16 genes. PTEN and p16 promoter methylation may be the main mechanism leading to the low expression of PTEN and p16 genes indicating the progress of tumor development. Our data suggest that a low PTEN and p16 expression due to methylation may contribute to the cancer progression and could be useful for prognosis of OSCC. Therefore, analysis of promoter methylation in such genes may provide a biomarker valuable for early detection of oral cancer.

Evaluation of cytotoxicity and genotoxicity of pesticide mixtures on lymphocytes.

The cytotoxicity and genotoxicity of pesticide mixtures viz. endosulfan + chlorpyrifos, chlorpyrifos + profenofos, and endosulfan + profenofos were evaluated on cultured human peripheral blood lymphocytes using assays for cell viability, and genotoxicity using chromosomal aberrations test and comet assay. The LC50 values for cytotoxicity were 3.50 µM, 4.18 µM, and 10.5 µM for profenofos, endosulfan, and chlorpyrifos respectively. When combined in equimolar concentrations, the LC50 values for cytotoxicity were 1.4 µM, 1.8 µM, and 2.0 µM for endosulfan + chlorpyrifos, chlorpyrifos + profenofos, and endosulfan + profenofos, respectively. Higher concentrations of individual pesticides (0.5-4.0 µM) but very low concentrations of pesticide mixtures caused significant DNA damage. Additive index values indicated a synergistic effect of toxicity for endosulfan + chlorpyrifos combination (1.12 TTU). The binary mixture of chlorpyrifos + profenofos showed an additive toxicity (0.46 TTU) while an antagonistic effect was observed for endosulfan + profenofos combination. Synergism could be due to these complementary pesticides simultaneously acting in different ways, magnifying their efficacy, whereas an additive interaction would imply that the chemicals are acting by the same mechanism and at the same target. Analysis of toxicity of pesticide mixtures may serve as important biomarker for occupational and household exposure to pesticides, with different modes of action.

Screening of phytochemicals against protease activated receptor 1 (PAR1), a promising target for cancer.

CONTEXT: Drug resistance and drug-associated toxicity are the primary causes for withdrawal of many drugs, although patient recovery is satisfactory in many instances. Interestingly, the use of phytochemicals in the treatment of cancer as an alternative to synthetic drugs comes with a host of advantages; minimum side effects, good human absorption and low toxicity to normal cells. Protease activated receptor 1 (PAR1) has been established as a promising target in many diseases including various cancers. Strong evidences suggest its role in metastasis also. OBJECTIVE: There are no natural compounds known to inhibit its activity, so we aimed to identify phytochemicals with antagonist activity against PAR1. METHODS: We screened phytochemicals from Naturally Occurring Plant-based Anticancer Compound-Activity-Target database (NPACT, http://crdd.osdd.net/raghava/npact/ ) against PAR1 using virtual screening workflow of Schrödinger software. It analyzes pharmaceutically relevant properties using Qikprop and calculates binding energy using Glide at three accuracy levels (high-throughput virtual screening, standard precision and extra precision). RESULTS AND CONCLUSION: Our study led to the identification of phytochemicals, which showed interaction with at least one experimentally determined active site residue of PAR1, showed no violations to Lipinski's rule of five along with predicted high human absorption. Furthermore, structural interaction fingerprint analysis indicated that the residues H255, D256, E260, S344, V257, L258, L262, Y337 and S344 may play an important role in the hydrogen bond interactions of the phytochemicals screened. Of these residues, H255 and L258 residues were experimentally proved to be important for antagonist binding. The residues Y183, L237, L258, L262, F271, L332, L333, Y337, L340, A349, Y350, A352, and Y353 showed maximum hydrophobic interactions with the phytochemicals screened. The results of this work suggest that phytochemicals Reissantins D, 24,25-dihydro-27-desoxywithaferin A, Isoguaiacin, 20-hydroxy-12-deoxyphorbol angelate, etc. could be potential antagonist of PAR1. However, further experimental studies are necessary to validate their antagonistic activity against PAR1.

Sequence-structure based phylogeny of GPCR Class A Rhodopsin receptors.

Current methods of G protein coupled receptors (GPCRs) phylogenetic classification are sequence based and therefore inappropriate for highly divergent sequences, sharing low sequence identity. In this study, sequence structure profile based alignment generated by PROMALS3D was used to understand the GPCR Class A Rhodopsin superfamily evolution using the MEGA 5 software. Phylogenetic analysis included a combination of Neighbor-Joining method and Maximum Likelihood method, with 1000 bootstrap replicates. Our study was able to identify potential ligand association for Class A Orphans and putative/unclassified Class A receptors with no cognate ligand information: GPR21 and GPR52 with fatty acids; GPR75 with Neuropeptide Y; GPR82, GPR18, GPR141 with N-arachidonylglycine; GPR176 with Free fatty acids, GPR10 with Tachykinin & Neuropeptide Y; GPR85 with ATP, ADP & UDP glucose; GPR151 with Galanin; GPR153 and GPR162 with Adrenalin, Noradrenalin; GPR146, GPR139, GPR142 with Neuromedin, Ghrelin, Neuromedin U-25 & Thyrotropin-releasing hormone; GPR171 with ATP, ADP & UDP Glucose; GPR88, GPR135, GPR161, GPR101with 11-cis-retinal; GPR83 with Tackykinin; GPR148 with Prostanoids, GPR109b, GPR81, GPR31with ATP & UTP and GPR150 with GnRH I & GnRHII. Furthermore, we suggest that this study would prove useful in re-classification of receptors, selecting templates for homology modeling and identifying ligands which may show cross reactivity with other GPCRs as signaling via multiple ligands play a significant role in disease modulation.

Epidermal growth factor receptor (EGFR) mutations as biomarker for head and neck squamous cell carcinomas (HNSCC).

CONTEXT: Mutations in tyrosine kinase domain (TK) of epidermal growth factor receptor (EGFR) lead to signalling interruptions in several cancers. OBJECTIVE: To understand EGFR mutations in head and neck squamous cell carcinomas (HNSCC), and their role as biomarkers. METHODS: Screened 129 HNSCC patients and 150 controls for mutations in the TK domain using polymerase chain reaction (PCR), single strand confirmatory polymorphism (SSCP) and sequencing. RESULTS: 81.39% of HNSCC had four mutations: G2155C, G2176A, C2188G and G2471A among these two mutations were also reported in other cancers where as two novel mutations are being reported for the first time in HNSCC. Mutational frequency was significantly associated with an advanced stage of HNSCC, habits of tobacco/alcohol and ages above 49 years. CONCLUSION: EGFR single nucleotide polymorphisms could be useful biomarkers of HNSCC.

Polymorphism of CYP3A4 2 and eNOS genes in the diabetic patients with hyperlipidemia undergoing statin treatment.

In individuals with diabetes, a log linear relationship exists between cholesterol levels and cardiovascular disease. Type 2 diabetes (T2D) and Statins, a cholesterol lowering drug, have a complex relationship. Statins are potent modulators of CYP3A4 2 enzyme and endothelial nitric oxide synthase (eNOS) functions in a number of cholesterol-independent, cardio protective actions in T2D. The aim of this study was to evaluate the CYP3A4 2 and eNOS gene mutations in a large number of T2D patients undergoing statin treatments. Blood samples were collected from 386 subjects in which 196 diabetic patients with hyperlipidemia were undergoing statin treatment (108 females and 88 males). The 190 healthy non-diabetic volunteers formed the control group. We investigated single nucleotide polymorphisms in diabetic patients and controls, and found that the statin therapy was not found to be effective in lowering LDL-cholesterol levels. Statistical analysis showed that T2D patients had significantly higher values of not only glucose levels but also a very high value of Triglycerides and cholesterol at the time of presentation. Our results for CYP3A4 2 showed that the genotype TT (wild type) had lower LDL when compared to TC (heterozygous). Similarly, the genotype TC (heterozygous) had lower LDL when compared to CC (homozygous). A similar trend was observed in the GG (wild type) and GT (heterozygous) of eNOS. In conclusion, we have described for the first time a significant correlation of statin treatment and CYP3A4 2 and eNOS gene polymorphisms in T2D, suggesting a new genetic susceptibility factor for insulin resistance and hyperlipidemia in T2D.

Pluripotent lineage of CD133 stem cells isolated from human skin samples.

Skin stem cells are very important in cosmetics, pharmacological and regenerative medicine and burn cases. Foreskin samples surgically removed after circumcision from boys below 7 years were collected and primary epidermal cells were prepared by enzymatic and mechanical tituration method. Selecting CD133 (prominin-1) multipotent stem cell marker, enriched stem cells were analyzed by MACS using CD133 antibodies conjugated with magnetic beads. CD133 positive and negative cells with specific skin stem cells markers like - CD34 (Universal stem cells marker), CD29 (integrin beta-1) and CD49f (integrin alpha-6) immunophenotypes were screened and sorted in flowcytometer. Further the expression of four embryonic genes or transcription factors of pluripotent stem cells were analyzed for pluripotent character of sorted cells. It was found that skin stem cell markers associated with CD133 cells, differentially expressed CD34, CD29 and CD49f immunophenotyes on both positive and negative CD133 cells in FACS analysis. The embryonic stem cell markers (induced pluripotent stem cell markers) like Oct4, SOX2, Notch-2 and K19 genes were expressed in CD133 positive epidermal cells. It is therefore evident that foreskin derived epidermal stem cells showed pluripotent or multipotent nature. This finding opens up avenues for new uses of these stem cells for direct cell seeding in wound healing, surgical suturing and drug screening.

Identification of Multi-kinase Allosteric Inhibitors of Oncogenic Targets EGFR1, PI3K, and BRAF Kinase.

AIM: This study aimed to identify promising allosteric inhibitors with the potential to inhibit EGFR1, PI3K, and BRAF kinases as a single agent or in a combination of existing drugs, thus acting as a therapeutic option when traditional drugs fail to give a beneficial response in disease pathology. BACKGROUND: Upregulation of EGFR1 activates several downstream signaling pathways, resulting in pathophysiological alterations that contribute to cancer. The RAS/RAF/MEK/ERK (MAPK) and PI3K/Akt/mTOR (PI3K/Akt/mTOR) pathways are major downstream signalling partners induced by EGFR1 activation. Despite their vast importance, allosteric FDA-approved drugs targeting EGFR1 and these pathways are not available. OBJECTIVE: The objective of the study is to identify novel multi-kinase small molecules with the potential to inhibit major sites of amplification of cancer signalling pathways, i.e., EGFR1, PI3K/Akt/mTOR, and RAS/RAF/MEK/ERK (MAPK) signalling pathways targeting allosteric sites. METHODS: In silico methods were used to identify the potential inhibitors using EGFR1, PI3, and BRAF crystal structures complexed with allosteric inhibitors. The potential novel molecules were confirmed for their drug-likeness. Their stability of binding was also confirmed using molecular dynamics simulation studies. To eliminate false negatives, this study used a pharmacophore and structure-based targeting method. RESULTS: The current study was effective in identifying drug-like small molecules, such as ZINC38783966, ZINC01456629, ZINC01456628, and 124173751, 137352549, 137353176, 137352399, 132020316 from ZINC and PubChem database, respectively, with a potential to bind EGFR1 (6DUK), PI3 (4A55) and BRAF (6P3D) at allosteric sites. A 50 ns molecular dynamics investigation also revealed that these potential novel multitarget kinase allosteric inhibitors exhibited stable binding. CONCLUSION: Alterations in EGFR1, PI3K/Akt/mTOR, and RAS/RAF/MEK/ERK (MAPK) signalling pathways are observed in cancers in high frequency and are also used by viral and environmental toxicants for pathologic purposes. These multi-kinase allosteric inhibitors will provide insight into allosteric drug discovery and deepen our understanding of targeting these pathways, either individually or in combination with orthosteric inhibitors.

Identification of novel allosteric binding sites and multi-targeted allosteric inhibitors of receptor and non-receptor tyrosine kinases using a computational approach.

EGFR1, VEGFR2, Bcr-Abl and Src kinases are key drug targets in non-small cell lung cancer (NSCLC), bladder cancer, pancreatic cancer, CML, ALL, colorectal cancer, etc. The available drugs targeting these kinases have limited therapeutic efficacy due to novel mutations resulting in drug resistance and toxicity, as they target ATP binding site. Allosteric drugs have shown promising results in overcoming drug resistance, but the discovery of allosteric drugs is challenging. The allosteric binding pockets are difficult to predict, as they are generally associated with high energy conformations and regulate protein function in yet unknown mechanisms. In addition, the discovery of drugs using conventional methods takes long time and goes through several challenges, putting the lives of many cancer patients at risk. Therefore, the aim of the present work was to apply the most successful, drug repurposing approach in combination with computational methods to identify kinase inhibitors targeting novel allosteric sites on protein structure and assess their potential multi-kinase binding affinity. Multiple crystal structures belonging to EGFR1, VEGFR2, Bcr-Abl and Src tyrosine kinases were selected, including mutated, inhibitor bound and allosteric conformations to identify potential leads, close to physiological conditions. Interestingly the potential inhibitors identified were peptides. The drugs identified in this study could be used in therapy as a single multi-kinase inhibitor or in a combination of single kinase inhibitors after experimental validation. In addition, we have also identified new hot spots that are likely to be druggable allosteric sites for drug discovery of kinase-specific drugs in the future.Communicated by Ramaswamy H. Sarma.

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.

Analysis of CCND1 protein and circulatory antioxidant enzyme activity association in oral squamous cell carcinoma.

Antioxidants are involved in the process of cellular damage prevention, which is considered as an avenue for cancer development. Free radicals are produced in the body upon exposure to stress, cigarette smoke, alcohol, toxins found in personal care products, pesticides in foods, radiation from the sun, viruses, germs or fungi etc. CCND1/CyclinD1 protein was found to be overexpressed in Oral squamous cell carcinoma. One hundred patients with oral squamous cell carcinoma were recruited along with hundred controls for this study from MNJ institute of Oncology with the approval of Ethics Committee, 5 ml blood samples were collected from each patient and centrifuged to collect serum for various assays. The antioxidant enzymes like catalase, SOD, GPX and GST were estimated using enzymatic assays. Results were expressed as unit of activity for mg of protein. Insilco analysis is performed using STRING v 11 Protein interaction tool. The patients with oral cancer had significantly reduced activities of SOD, GST and GPX (1.49 ± 0.49, 3.97 ± 0.86 and 10.7 ± 0.73 respectively) compared to healthy controls (4.37 ± 1.43, 6.10 ± 1.12 and 13.8 ± 1.25 respectively) (p < 0.005). However no significant difference was observed with regard to catalase activity (2.71 ± 6.51 and 4.03 ± 1.48) (p = 0.28). The proteins interaction PPI enrichment p-value was found to be 3.22e-10 predicted significantly more interactions. Our research findings shown that there was a decline in activity of superoxide dismutase, glutathione peroxidase and glutathione s transferase in addition, personal habits like smoking play a major role in the development and progression of oral carcinogenesis and based on Insilco analysis results CCND1/Cyclin D1 could be the potential therapeutic target in oral squamous cell carcinoma.

Saudi Familial Hypercholesterolemia Patients With Rare LDLR Stop Gain Variant Showed Variable Clinical Phenotype and Resistance to Multiple Drug Regimen.

Familial hypercholesterolemia (FH), a well-known lipid disease caused by inherited genetic defects in cholesterol uptake and metabolism is underdiagnosed in many countries including Saudi Arabia. The present study aims to identify the molecular basis of severe clinical manifestations of FH patients from unrelated Saudi consanguineous families. Two Saudi families with multiple FH patients fulfilling the combined FH diagnostic criteria of Simon Broome Register, and the Dutch Lipid Clinic Network (DLCN) were recruited. LipidSeq, a targeted resequencing panel for monogenic dyslipidemias, was used to identify causative pathogenic mutation in these two families and in 92 unrelated FH cases. Twelve FH patients from two unrelated families were sharing a very rare, pathogenic and founder LDLR stop gain mutation i.e., c.2027delG (p.Gly676Alafs*33) in both the homozygous or heterozygous states, but not in unrelated patients. Based on the variant zygosity, a marked phenotypic heterogeneity in terms of LDL-C levels, clinical presentations and resistance to anti-lipid treatment regimen (ACE inhibitors, ß-blockers, ezetimibe, statins) of the FH patients was observed. This loss-of-function mutation is predicted to alter the free energy dynamics of the transcribed RNA, leading to its instability. Protein structural mapping has predicted that this non-sense mutation eliminates key functional domains in LDLR, which are essential for the receptor recycling and LDL particle binding. In conclusion, by combining genetics and structural bioinformatics approaches, this study identified and characterized a very rare FH causative LDLR pathogenic variant determining both clinical presentation and resistance to anti-lipid drug treatment.

Molecular & biochemical analysis of Pro12Ala variant of PPAR-γ2 gene in type 2 diabetes mellitus.

Diabetes has emerged as a major threat to human life globally. Genomic studies have found a significant link between the Pro12Ala polymorphism of the PPAR-γ2 gene with incidence as well as occurrence of the risk of metabolic syndrome. The present study was aimed at assessing the PPAR-γ2 variant in an Asian Indian cohort of type 2 diabetes patients and its correlation with metabolic parameters. The present case-control study involved 100 type 2 diabetic patients and 100 asymptomatic healthy volunteers enrolled in random. Assessment of demographic factors and biochemical parameters were done for all enrolled. In addition, genotyping for the Pro12Ala (CCA to GCA) polymorphism was done by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) technology. The genotyping study detected the frequency of the CC genotype (Pro12Pro) to be higher in frequency in comparison to the heterozygous CG genotype in both, cases and controls. The homozygous GG genotype (Ala12Ala) was not detected in any of the cases or controls assessed. Biochemical analysis of the levels of malondialdehyde (MDA) detected a significant increase (p < 0.0001). Additionally, increase in levels of fasting and postprandial glucose, total cholesterol, triglycerides, and parameters of the liver and renal function tests were detected. This study detected the PPAR-γ2 to be a significant biomarker for type 2 diabetes mellitus.

Polymorphisms in MGP gene and their association with lead toxicity.

Matrix gamma-carboxy glutamic acid protein (MGP) is a 10-kDa secreted protein containing five residues of the vitamin K-dependent calcium binding amino acid gamma-carboxyglutamic acid (Gla). This study was carried out to examine the effects of MGP gene promoter polymorphism (T-138C) on blood lead levels (BLL) and hematological parameters in 113 battery manufacturing unit workers occupationally exposed to lead and 102 controls. Genotypes for the MGP T-138C polymorphism were determined by PCR and restriction fragment length digestion. BLL were determined by Anode Stripping Voltammetry using ESA Model 3010B Lead analyzer. Complete blood picture (CBP) was analyzed using ADVIA Cell counter for each sample. The frequencies of MGP-TT, CT and CC genotypes in our population were 38.6%, 44.3%, and 17.2%, respectively. The frequencies for T and C alleles were 0.612 and 0.386, respectively. Although BLL did not differ significantly among genotypes; they were higher in workers with TT/CT genotype compared to CC genotype subjects (76-88 microg/dL vs 22-45 microg/dL, p > 0.05). About 29.2% of volunteers (n = 33) from the occupationally exposed group had hemoglobin levels below 10.0 gms/dl. There was no significant difference in total white cell count and platelet count between occupational and non-exposed groups. The possible role of SNPs in the promoter region of MGP gene with relation to lead toxicity was investigated for the first time in the Indian population; although significance could not be achieved in this study, further assessments over a larger population size may help in better understanding of the consequences of lead exposure.

Role of cytochrome epoxygenase (CYP2J2) in the pathophysiology of coronary artery disease in South Indian population.

BACKGROUND: The cytochrome P-450 2J2 (CYP2J2) is known to be one of the major enzymes of epoxygenase pathway of arachidonic acid in extrahepatic tissues, which produces series of regioisomeric cis-epoxyeicosatrienoic acids (EETs) such as 5,6-, 8,9-, 11,12-, and 14,15-EETs. In the present study, we analyzed the impact of a genetic variant in CYP2J2 on coronary artery disease (CAD) in the Telangana region of Indian population. MATERIAL AND METHODS: The case-control study consisted of 100 CAD cases and 110 healthy controls. The deoxyribonucleic acid was extracted using the salting out method. Genotyping and gene expression was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism and real-time-PCR methods. RESULTS: In the present study, the percentage of smokers, alcoholics, hypertensive patients, and diabetics was high. Increase in fasting glucose, urea, creatinine, fasting triglycerides, total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), total cholesterol/high-density lipoprotein (TC/HDL), LDL/HDL, homocysteine, and C-reactive protein levels were significantly higher in patients with CAD than in controls (p < 0.001). CYP2J2 G-50T was associated with CAD (p = 0.04). The mRNA expression of CYP2J2 showed altered gene expression in this study among CAD patients in comparison with control (p = 0.01). CONCLUSIONS: A functionally relevant polymorphism of the CYP2J2 gene was independently associated with an increased risk of CAD.

Determination of AChE levels and genotoxic effects in farmers occupationally exposed to pesticides.

Pesticides can cause cytogenetic effects and lower the acetyl cholinesterase (AChE) levels in farmers exposed to pesticides. In this study, 210 farmers exposed to pesticides and 160 non-exposed individuals were enrolled for determining the genotoxicity and AChE levels. The AChE levels were determined in plasma and RBC lysate from blood samples collected from farmers and control subjects. AChE (true and pseudo) estimation done by the colorimetric method revealed that there was a progressive fall in both the RBC and plasma AChE levels in exposed individuals compared to unexposed individuals, which correlated with the severity of exposure (253.5 versus 311.1 and 142.3 versus 152.1; P < 0.001). Cytogenetic studies showed an increase in DNA damage and higher chromosomal aberrations (CAs) in exposed farmers compared to the control subjects (26.13 versus 07.61 and 21.37 versus 1.52; P < 0.001). When comparing the AChE levels with DNA damage and structural CA frequencies, there was a negative linear correlation. Therefore based on these findings, it is concluded that genotoxic biomarkers like CA frequencies, DNA damage data along with AChE levels are important parameters for determining farmer's health who are exposed to pesticides in any situation.

Promoter Hypermethylation of the ATM Gene as a Novel Biomarker for Breast Cancer

Background: Breast cancer may be induced by activation of protooncogenes to oncogenes and in many cases inactivation of tumor suppressor genes. Ataxia telangiectasia mutated (ATM) is an important tumor suppressor gene which plays central roles in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of double-strand breaks of DNA. In breast cancer, decrease ATM expression correlates with a poor outcome; however, the molecular mechanisms underlying downregulation are still unclear. Promoter hypermethylation may contribute in downregulation. Hence the present investigation was designed to evaluate promoter methylation and expression of the ATM gene in breast cancer cases, and to determine links with clinical and demographic manifestations, in a South Indian population. Methods: Tumor biopsy samples were collected from 50 pathologically confirmed sporadic breast cancer cases. DNA was isolated from tumor and adjacent non-tumorous regions, and sodium bisulfite conversion and methylation-specific PCR were performed using MS-PCR primers for the ATM promoter region. In addition, ATM mRNA expression was also analyzed for all samples using real-time PCR. Results: Fifty eight percent (58%) of cancer tissue samples showed promoter hypermethylation for the ATM gene, in contrast to only 4.44% of normal tissues (p= 0.0001). Furthermore, ATM promoter methylation was positively associated with age (p = 0.01), tumor size (p=0.045) and advanced stage of disease i.e. stages III and IV (p =0.019). An association between promoter hypermethylation and lower expression of ATM mRNA was also found (p=0.035). Conclusion: We report for the first time that promoter hypermethylation of ATM gene may be useful as a potential new biomarker for breast cancer, especially in the relatively young patients.

Pharmacodynamic and cytogenetic evaluation in CYP2C19*2 and CYP2C19*3 allelomorphism in South Indian population with clopidogrel therapy.

BACKGROUND: Genetic factors play a significant role in pathogenesis of most diseases of heart. The present study was undertaken to correlate coronary artery disease with demographical, biochemical alterations, SNPs, gene expression and chromosomal abnormalities and for further enlightening the investigation in this field. METHODS: 150 patients taking clopidogrel drug were selected and single nucleotide polymorphism was done by PCR-RFLP techniques. With the same patients cytogenetic analysis was carried out on leukocyte cultures by karyotyping. Gene expression studies for 20 CAD patients and normal controls were done by RT-PCR techniques. RESULTS: In this study of patients with coronary artery disease the frequencies of the Extreme Metabolizers, Intermediate Metabolizers in CYP2C19*2 (rs4244285) were present in 90% and 10% but no Poor Metabolizers were found in this allele. The frequencies of Extreme Metabolizer, Intermediate Metabolizer and Poor Metabolizer in CYP2C19*3 (rs4986893) were present in 41%, 50% and 9% respectively. Among 20 CAD samples, 13 of 20 (65%) showed CYP2C19 gene over expression in CAD patients and all controls showed normal expression. Among the 150 CAD patients, 145 had normal karyotype, only five patients showed change in normal karyogram carried out by leukocyte culture. CONCLUSION: Genetic testing of CYP2C19 may help in prescribing a dose according to genetic makeup and represent the initial steps towards the development of diagnostic tests and therapeutic strategies that will substantially improve human health. This study highlights the progress that has been made in using pharmacogenomic and gene expression analysis, cardiovascular genomic research and the potential for applying these findings in clinical medicine.