Aberrant epigenetic inactivation of RASSF1A and MGMT gene and genetic mutations of KRAS, cKIT and BRAF in Indian testicular germ cell tumours.

Testicular germ cell tumor (TGCT) development may involve a series of modification at epigenetic and genetic level which may act synergistically and transform the primordial gonocyte. This study evaluated the frequency and distribution pattern of RASSF1A/MGMT gene methylation and KRAS, BRAF and cKIT gene mutation in Indian TGCT patient, and their correlation with clinicopathological features. Forty-one TGCT tumors were used to investigate hypermethylation of RASSF1A and MGMT gene and mutations of KRAS codon 12/13, BRAF V600E and cKIT exon 17 mutations. RASSF1A and MGMT methylation was noted in 58.5% and 10% of the TGCTs. A higher frequency of RASSF1A methylation was noted in seminomas (71%, 17/24), while MGMT methylation was frequent in mixed tumors (23%, 3/13). Interestingly, 3 of 41 cases showed concurrent methylation of both the genes. The absence of tumor necrosis was significantly associated with increased frequency of MGMT hypermethylation (30% vs. 3%, p = 0.03). KRAS mutation was identified in 17% (7/41), while none showed BRAF and cKIT mutation. KRAS mutations were predominantly found in codon 12 with G12V as the most recurrent mutations. Mixed germ tumors tends to show increased frequency of KRAS mutation (31%, 4/13), followed by pure seminomas (4%, 1/24). Interestingly, KRAS mutation rate was significantly higher in metastatic tumors in comparison to primary tumors (100% vs. 13%, p = 0.02). No other association of RASSF1A/MGMT/KRAS alterations with other clinicopathological features was noted. In conclusion, these data support the notion that the cancer-associated alterations in the RASSF1, MGMT and KRAS gene may significantly contribute to TGCT pathogenesis.

Molecular evaluation of BRAF V600 mutation and its association with clinicopathological characteristics: First findings from Indian malignant melanoma patients.

Mutations in the BRAF gene have been described to occur in two-third of melanomas. The objective of the study was to establish the frequency of BRAF V600E/K/R mutation in a series of melanomas from Indian origin and to correlate mutation status with clinicopathological features. Seventy melanoma cases were evaluated for BRAF V600 mutation by pyrosequencing. Overall, BRAF mutations were detected in 30% of the patients. All mutations observed were missense type (GTG > GAG) resulting in p.V600E, while none showed V600K/R mutation. The frequency of BRAF V600E mutations were more in patients with onset age of 50 years. BRAF mutations were significantly associated with tumor site wherein more mutations were seen in tumors from head and neck and extremities region. Acral and mucosal tumor subtype showed a mutation frequency of 31% and 20%, respectively. Epithelial cell morphology tends to harbor frequent BRAF V600E mutation (36%) than other morphological subtypes. Tumors with ulceration and necrosis showed increased BRAF mutation rate (32.5% and 33%) respectively. In conclusion, this is the first study to report a mutation frequency of 30% in this cohort. Our results demonstrated that the BRAF V600E mutation is a frequent event in Indian melanomas, and represents an important molecular target for novel therapeutic approaches.

Association of CYBA G640A variation with coronary artery disease in Indians.

INTRODUCTION: Oxidative stress induces atherosclerosis by triggering an inflammatory cascade within the vascular wall. OBJECTIVE: To investigate the role of pro-oxidant and antioxidant gene variations with CAD in Indian subjects. MATERIALS & METHODS: It's a case-control study and genotyping for the variants MPO G-463A, CYBA G640A, SOD2 Val16Ala and CAT C-262T were performed by conventional PCR techniques. RESULTS: Only CYBA G640A variant allele was found to be significantly (p = 0.0075) associated with CAD. CONCLUSION: Although CYBA G640A variation was found to be significant, a larger study is needed to validate these results and establish its role as a biomarker.

Effect of CYP2C9 and VKORC1 genetic variations on warfarin dose requirements in Indian patients.

BACKGROUND: Warfarin, an oral anticoagulant is used in patients who are at increased risk of developing blood clots. The management of warfarin therapy is challenging because it shows large inter and intra individual variability in patient response due to factors like age, gender, diet, concurrent drug interactions and variations in CYP2C9 and VKORC1 genes. Studies implicate that polymorphisms in VKORC1 and CYP2C9 genes are associated with reduced doses of warfarin. The aim of our current study was to characterize the effects of VKORC1 and CYP2C9 gene variations that contribute to variability in warfarin dosing in Indian patients. METHODS: Genomic DNA was extracted from 103 patients undergoing warfarin therapy. Their mean daily warfarin dose, INR and demographics were recorded and genotyping of VKORC1 and CYP2C9 gene was performed by PCR-RFLP method. RESULTS: Individuals with wild type genotypes required highest mean warfarin dosage of 4.72 mg/day while VKORC1 variants required 3.6 mg/day to maintain their therapeutic INR. CYP2C9*2 genotype was not found to affect the warfarin maintenance dosages. The odds ratio for developing supra therapeutic INR in patients carrying VKORC1 variant allele when compared to wild types was 13.96 (95% CI; 4.85 - 44.65. Other factors affecting warfarin dosages were age and weight. CONCLUSION: Inclusion of pharmacogenetic data along with clinical parameters would help better predict warfarin doses in Indian patients.

Distinct endothelial phenotypes evoked by arterial waveforms derived from atherosclerosis-susceptible and -resistant regions of human vasculature.

Atherosclerotic lesion localization to regions of disturbed flow within certain arterial geometries, in humans and experimental animals, suggests an important role for local hemodynamic forces in atherogenesis. To explore how endothelial cells (EC) acquire functional/dysfunctional phenotypes in response to vascular region-specific flow patterns, we have used an in vitro dynamic flow system to accurately reproduce arterial shear stress waveforms on cultured human EC and have examined the effects on EC gene expression by using a high-throughput transcriptional profiling approach. The flow patterns in the carotid artery bifurcations of several normal human subjects were characterized by using 3D flow analysis based on actual vascular geometries and blood flow profiles. Two prototypic arterial waveforms, "athero-prone" and "athero-protective," were defined as representative of the wall shear stresses in two distinct regions of the carotid artery (carotid sinus and distal internal carotid artery) that are typically "susceptible" or "resistant," respectively, to atherosclerotic lesion development. These two waveforms were applied to cultured EC, and cDNA microarrays were used to analyze the differential patterns of EC gene expression. In addition, the differential effects of athero-prone vs. athero-protective waveforms were further characterized on several parameters of EC structure and function, including actin cytoskeletal organization, expression and localization of junctional proteins, activation of the NF-kappaB transcriptional pathway, and expression of proinflammatory cytokines and adhesion molecules. These global gene expression patterns and functional data reveal a distinct phenotypic modulation in response to the wall shear stresses present in atherosclerosis-susceptible vs. atherosclerosis-resistant human arterial geometries.

Improving the statistical detection of regulated genes from microarray data using intensity-based variance estimation.

BACKGROUND: Gene microarray technology provides the ability to study the regulation of thousands of genes simultaneously, but its potential is limited without an estimate of the statistical significance of the observed changes in gene expression. Due to the large number of genes being tested and the comparatively small number of array replicates (e.g., N = 3), standard statistical methods such as the Student's t-test fail to produce reliable results. Two other statistical approaches commonly used to improve significance estimates are a penalized t-test and a Z-test using intensity-dependent variance estimates. RESULTS: The performance of these approaches is compared using a dataset of 23 replicates, and a new implementation of the Z-test is introduced that pools together variance estimates of genes with similar minimum intensity. Significance estimates based on 3 replicate arrays are calculated using each statistical technique, and their accuracy is evaluated by comparing them to a reliable estimate based on the remaining 20 replicates. The reproducibility of each test statistic is evaluated by applying it to multiple, independent sets of 3 replicate arrays. Two implementations of a Z-test using intensity-dependent variance produce more reproducible results than two implementations of a penalized t-test. Furthermore, the minimum intensity-based Z-statistic demonstrates higher accuracy and higher or equal precision than all other statistical techniques tested. CONCLUSION: An intensity-based variance estimation technique provides one simple, effective approach that can improve p-value estimates for differentially regulated genes derived from replicated microarray datasets. Implementations of the Z-test algorithms are available at http://vessels.bwh.harvard.edu/software/papers/bmcg2004.