Puthiya M. Gopinath

Genome-wide analysis correlates Ayurveda Prakriti

The practice of Ayurveda, the traditional medicine of India, is based on the concept of three major constitutional types (Vata, Pitta and Kapha) defined as “Prakriti”. To the best of our knowledge, no study has convincingly correlated genomic variations with the classification of Prakriti. In the present study, we performed genome-wide SNP (single nucleotide polymorphism) analysis (Affymetrix, 6.0) of 262 well-classified male individuals (after screening 3416 subjects) belonging to three Prakritis. We found 52 SNPs (p ≤ 1 × 10−5) were significantly different between Prakritis, without any confounding effect of stratification, after 106 permutations. Principal component analysis (PCA) of these SNPs classified 262 individuals into their respective groups (Vata, Pitta and Kapha) irrespective of their ancestry, which represent its power in categorization. We further validated our finding with 297 Indian population samples with known ancestry. Subsequently, we found that PGM1 correlates with phenotype of Pitta as described in the ancient text of Caraka Samhita, suggesting that the phenotypic classification of India’s traditional medicine has a genetic basis; and its Prakriti-based practice in vogue for many centuries resonates with personalized medicine.

DNA methylation analysis of phenotype specific stratified Indian population

BackgroundDNA methylation and its perturbations are an established attribute to a wide spectrum of phenotypic variations and disease conditions. Indian traditional system practices personalized medicine through indigenous concept of distinctly descriptive physiological, psychological and anatomical features known as prakriti. Here we attempted to establish DNA methylation differences in these three prakriti phenotypes.MethodsFollowing structured and objective measurement of 3416 subjects, whole blood DNA of 147 healthy male individuals belonging to defined prakriti (Vata, Pitta and Kapha) between the age group of 20-30years were subjected to methylated DNA immunoprecipitation (MeDIP) and microarray analysis. After data analysis, prakriti specific signatures were validated through bisulfite DNA sequencing.ResultsDifferentially methylated regions in CpG islands and shores were significantly enriched in promoters/UTRs and gene body regions. Phenotypes characterized by higher metabolism (Pitta prakriti) in individuals showed distinct promoter (34) and gene body methylation (204), followed by Vata prakriti which correlates to motion showed DNA methylation in 52 promoters and 139 CpG islands and finally individuals with structural attributes (Kapha prakriti) with 23 and 19 promoters and CpG islands respectively. Bisulfite DNA sequencing of prakriti specific multiple CpG sites in promoters and 5′-UTR such as; LHX1 (Vata prakriti), SOX11 (Pitta prakriti) and CDH22 (Kapha prakriti) were validated. Kapha prakriti specific CDH22 5′-UTR CpG methylation was also found to be associated with higher body mass index (BMI).ConclusionDifferential DNA methylation signatures in three distinct prakriti phenotypes demonstrate the epigenetic basis of Indian traditional human classification which may have relevance to personalized medicine.

Determinants of Prakriti, the Human Constitution Types of Indian Traditional Medicine and its Correlation with Contemporary Science

Background: Constitutional type of an individual or prakriti is the basic clinical denominator in Ayurveda, which defines physical, physiological, and psychological traits of an individual and is the template for individualized diet, lifestyle counseling, and treatment. The large number of phenotype description by prakriti determination is based on the knowledge and experience of the assessor, and hence subject to inherent variations and interpretations. Objective: In this study we have attempted to relate dominant prakriti attribute to body mass index (BMI) of individuals by assessing an acceptable tool to provide the quantitative measure to the currently qualitative ayurvedic prakriti determination. Materials and Methods: The study is cross sectional, multicentered, and prakriti assessment of a total of 3416 subjects was undertaken. Healthy male, nonsmoking, nonalcoholic volunteers between the age group of 20-30 were screened for their prakriti after obtaining written consent to participate in the study. The prakriti was determined on the phenotype description of ayurvedic texts and simultaneously by the use of a computer-aided prakriti assessment tool. Kappa statistical analysis was employed to validate the prakriti assessment and Chi-square, Cramer′s V test to determine the relatedness in the dominant prakriti to various attributes. Results: We found 80% concordance between ayurvedic physician and software in predicting the prakriti of an individual. The kappa value of 0.77 showed moderate agreement in prakriti assessment. We observed a significant correlations of dominant prakriti to place of birth and BMI with Chi-square, P < 0.01 (Cramer′s V-value of 0.156 and 0.368, respectively). Conclusion: The present study attempts to integrate knowledge of traditional ayurvedic concepts with the contemporary science. We have demonstrated analysis of prakriti classification and its association with BMI and place of birth with the implications to one of the ways for human classification.

Intraindividual somatic variations in MTHFR gene polymorphisms in relation to colon cancer

AIM MTHFR mediates the one carbon metabolism pathway. Two common genetic variants, C677T and A1298C, of MTHFR are associated with number of human diseases, including cancer, as well as being involved in the modulation of therapy outcome to antifolate drugs. To understand the distribution pattern of SNPs among different tissues of an individual, we examined MTHFR polymorphisms in normal and colon cancer tissues and compared the genotype frequencies in peripheral blood samples. MATERIALS & METHODS DNA was isolated from tumor tissue and matched normal tissues from 155 colon cancer patients. These samples as well as DNA from blood samples of the control group (n = 294) were analyzed for MTHFR polymorphisms by PCR-RFLP and confirmed by a direct DNA sequencing method. RESULTS Our data suggest that the allele and genotype frequencies of C677T and A1298C were significantly different between tumor tissues and both types of normal tissues. We have established that MTHFR variants that exist in tumor and matched normal tissues of colon cancer patients differ suggesting somatic variation in MTHFR polymorphisms among different tissues of an individual. The MTHFR A1298C polymorphism was associated with risk of colon cancer. CONCLUSION Different MTHFR variants may exist in different tissues to maintain physiological functions and may have implications for disease susceptibility and pharmacogenomics based therapies. Original submitted 21 January 2013; Revision submitted 3 January 2014.

Upregulation of TFAM and mitochondria copy number in human lymphoblastoid cells

Mitochondria are central to several physiological and pathological conditions in humans. In the present study, we performed copy number analysis of nuclear encoded mitochondrial genes, in peripheral blood mononuclear cells (PBMCs) and its representative lymphoblastoid cells (LCLs). We have observed hyper diploid copies of mitochondrial transcription factor A (TFAM) gene in the LCLs along with increased mtDNA copy number, mitochondrial mass, intracellular ROS and mitochondrial membrane potential, suggesting elevated mitochondrial biogenesis in LCLs. Gene expression analysis confirmed TFAM over-expression in LCLs when compared to PBMC. Based on our observation, we suggest that increased copy number of TFAM gene upregulates its expression, increases mtDNA copy numbers and protects it from oxidative stress induced damage in the transformed LCLs.

Prevalence of human papillomavirus types and phylogenetic analysis of HPV-16 L1 variants from Southern India.

BACKGROUND The human papillomavirus (HPV) and its variants show wide geographical distribution and have been reported to cause cervical lesions. With cervical neoplasia as the leading cancer in Indian women, the aim of the present study was to evaluate the multiple infection HPV type distribution and variant genotypes in cervical samples from the coastal Karnataka region, India. MATERIALS AND METHODS A total of 212 samples were screened by nested polymerase chain reaction using PGMY9/11 and GP5+/6+ primers. HPV positive samples were sequenced to identify the types and a phylogenetic tree was constructed using the neighbor-joining method. RESULTS Sequence analysis identified a total of 14 HPV types distributed in 20%, 73.3% and 82.5% of non-malignant, pre-malignant [low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL)] and cervical cancer samples. The distribution of high risk HPV in cancer samples was HPV 16, 76.4%, HPV18, 11.7%, HPV81, 2.9%, HPV31, 1.4%, HPV35, 1.4% and HPV 45, 1.4%. Multiple infections were observed in 11.8% of tumor samples with HPV 16 contributing to 62.5% of cases. In non-malignant samples, 20% of HPV positive samples were detected with HPV16, 82.3%, HPV33, 5.8% and HPV58, 5.8% and very low incidence of multiple infections. Comparative phylogenetic analysis of HPV variants identified 9 HPV sequences as new papillomavirus species, predominantly classified as European lineage type. CONCLUSIONS The findings for HPV infections associated with progression of cervical cancer in coastal Karnataka region and HPV variant analysis provide baseline data for prevention and HPV vaccination programs.

A novel gene THSD7A is associated with obesity.

Body mass index (BMI) is a non-invasive measurement of obesity. It is commonly used for assessing adiposity and obesity-related risk prediction. Genetic differences between ethnic groups are important factors, which contribute to the variation in phenotypic effects. India inhabited by the first out-of-Africa human population and the contemporary Indian populations are admixture of two ancestral populations; ancestral north Indians (ANI) and ancestral south Indians (ASI). Although ANI are related to Europeans, ASI are not related to any group outside Indian-subcontinent. Hence, we expect novel genetic loci associated with BMI. In association analysis, we found eight genic SNPs in extreme of distribution (P⩽3.75 × 10−5), of which WWOX has already been reported to be associated with obesity-related traits hence excluded from further study. Interestingly, we observed rs1526538, an intronic SNP of THSD7A; a novel gene significantly associated with obesity (P=2.88 × 10−5, 8.922 × 10−6 and 2.504 × 10−9 in discovery, replication and combined stages, respectively). THSD7A is neural N-glycoprotein, which promotes angiogenesis and it is well known that angiogenesis modulates obesity, adipose metabolism and insulin sensitivity, hence our result find a correlation. This information can be used for drug target, early diagnosis of obesity and treatment.

Comprehensive DNA copy number profile and BAC library construction of an Indian individual

Bacterial artificial chromosomes (BACs) are used in genomic variation studies due to their capacity to carry a large insert, their high clonal stability, low rate of chimerism and ease of manipulation. In the present study, an attempt was made to create the first genomic BAC library of an anonymous Indian male (IMBL4) consisting of 100,224 clones covering the human genome more than three times. Restriction mapping of 255 BAC clones by pulse field gel electrophoresis confirmed an average insert size of 120 kb. The library was screened by PCR using SHANK3 (SH3 and multiple ankyrin repeat domains 3) and OLFM3 (olfactomedin 3) specific primers. A selection of clones was analyzed by fluorescent in situ hybridization (FISH) and sequencing. Fine mapping of copy number variable regions by array based comparative genomic hybridization identified 467 CNVRs in the IMBL4 genome. The IMBL4 BAC library represents the first cataloged Indian genome resource for applications in basic and clinical research.

Effect of Amalaki rasayana on DNA damage and repair in randomized aged human individuals

ETHNOPHARMACOLOGICAL RELEVANCE Preparations from Phyllanthus emblica called Amalaki rasayana is used in the Indian traditional medicinal system of Ayurveda for healthy living in elderly. The biological effects and its mechanisms are not fully understood. Since the diminishing DNA repair is the hallmark of ageing, we tested the influence of Amalaki rasayana on recognized DNA repair activities in healthy aged individuals. METHODS Amalaki rasayana was prepared fresh and healthy aged randomized human volunteers were administrated with either rasayana or placebo for 45 days strictly as per the traditional text. The DNA repair was analyzed in peripheral blood mononuclear cells before and after rasayana administration and after 45 days post-rasayana treatment regimen. UVC-induced DNA strand break repair (DSBR) based on extent of DNA unwinding by fluorometric analysis, nucleotide excision repair (NER) by flow cytometry and constitutive base excision repair (BER) by gap filling method were analyzed. RESULTS Amalaki rasayana administration stably maintained/enhanced the DSBR in aged individuals. There were no adverse side effects. Further, subjects with different body mass index showed differential DNA strand break repair capacity. No change in unscheduled DNA synthesis during NER and BER was observed between the groups. CONCLUSION Intake of Amalaki rasayana by aged individuals showed stable maintenance of DNA strand break repair without toxic effects. However, there was no change in nucleotide and base excision repair activities. Results warrant further studies on the effects of Amalaki rasayana on DSBR activities.

Modulatory action of 2-deoxy-D-glucose on mitomycin C-and 4-nitroquinoline-1-oxide-induced genotoxicity in Swiss albino mice in vivo.

BACKGROUND 2-Deoxy-D-glucose (2-DG), a structural analog of glucose is an effective inhibitor of glucose metabolism and ATP production. It selectively accumulates in cancer cells and interferes with glycolysis leading to cell death. 2-DG is shown to differentially enhance the radiation-induced damage in cancer cells both under euoxic and hypoxic conditions. A combination of 2-DG and ionizing radiation selectively destroys tumors while protecting the normal tissue. 2-DG is being advocated as an adjuvant in the radiotherapy and chemotherapy of cancer. OBJECTIVE The present investigation focuses on the modulatory effect of 2-DG on mitomycin C- (MMC) and 4-nitroquinoline-1-oxide (4-NQO)-induced cytogenetic damage in bone marrow cells of Swiss albino mice in vivo. MATERIALS AND METHODS Experimental animals were pretreated with 2-DG (500 mg/kg, i.p.) for five consecutive days followed by MMC (2 mg/kg, i.p) or 4-NQO (15 mg/kg, i.p.), 24 h prior to sacrifice. Control animals were given either the mixture of olive oil and acetone (3:1) or distilled water. Bone marrow cells were processed for the micronucleus assay and metaphase analysis for estimating cytogenetic damage. RESULTS 2-DG significantly (P < 0.001) reduced the frequency of aberrant cells induced by MMC (approximately 90%) and 4-NQO (approximately 74%). Incidence of micronucleated polychromatic erythrocytes (MnPCEs) induced by the mutagens were reduced up to 68%. CONCLUSION 2-DG effectively reduces the MMC-and 4-NQO-induced genotoxicity.