Do environmentally induced DNA variations mediate adaptation in Aspergillus flavus exposed to chromium stress in tannery sludge?

BACKGROUND: Environmental stress induced genetic polymorphisms have been suggested to arbitrate functional modifications influencing adaptations in microbes. The relationship between the genetic processes and concomitant functional adaptation can now be investigated at a genomic scale with the help of next generation sequencing (NGS) technologies. Using a NGS approach we identified genetic variations putatively underlying chromium tolerance in a strain of Aspergillus flavus isolated from a tannery sludge. Correlation of nsSNPs in the candidate genes (n = 493) were investigated for their influence on protein structure and possible function. Whole genome sequencing of chromium tolerant A. flavus strain (TERIBR1) was done (Illumina HiSeq2000). The alignment of quality trimmed data of TERIBR1 with reference NRRL3357 (accession number EQ963472) strain was performed using Bowtie2 version 2.2.8. SNP with a minimum read depth of 5 and not in vicinity (10 bp) of INDEL were filtered. Candidate genes conferring chromium resistance were selected and SNPs were identified. Protein structure modeling and interpretation for protein-ligand (CrO4- 2) docking for selected proteins harbouring non-synonymous substitutions were done using Phyre2 and PatchDock programs. RESULTS: High rate of nsSNPs (approximately 11/kb) occurred in selected candidate genes for chromium tolerance. Of the 16 candidate genes selected for studying effect of nsSNPs on protein structure and protein-ligand interaction, four proteins belonging to the Major Facilitator Superfamily (MFS) and recG protein families showed significant interaction with chromium ion only in the chromium tolerant A. flavus strain TERIBR1. CONCLUSIONS: Presence of nsSNPs and subsequent amino-acid alterations evidently influenced the 3D structures of the candidate proteins, which could have led to improved interaction with (CrO4- 2) ion. Such structural modifications might have enhanced chromium efflux efficiency of A. flavus (TERIBR1) and thereby offered the adaptation benefits in counteracting chromate stress. Our findings are of fundamental importance to the field of heavy-metal bio-remediation.

Phylogenetic analyses reveal molecular signatures associated with functional divergence among Subtilisin like Serine Proteases are linked to lifestyle transitions in Hypocreales.

BACKGROUND: Subtilisin-like serine proteases or Subtilases in fungi are important for penetration and colonization of host. In Hypocreales, these proteins share several properties with other fungal, bacterial, plant and mammalian homologs. However, adoption of specific roles in entomopathogenesis may be governed by attainment of unique biochemical and structural features during the evolutionary course. Due to such functional shifts Subtilases coded by different family members of Hypocreales acquire distinct features according to respective hosts and lifestyle. We conducted phylogenetic and DIVERGE analyses and identified important protein residues that putatively assign functional specificity to Subtilases in fungal families/species under the order Hypocreales. RESULTS: A total of 161 Subtilases coded by 10 species from five different families under the fungal order Hypocreales was included in the analysis. Based on the presence of conserved domains, the Subtilase genes were divided into three subfamilies, Subtilisin (S08.005), Proteinase K (S08.054) and Serine-carboxyl peptidases (S53.001). These subfamilies were investigated for phylogenetic associations, protein residues under positive selection and functional divergence among paralogous clades. The observations were co-related with the life-styles of the fungal families/species. Phylogenetic and Divergence analyses of Subtilisin (S08.005) and Proteinase K (S08.054) families of proteins revealed that the paralogous clades were clear-cut representation of familial origin of the protein sequences. We observed divergence between the paralogous clades of plant-pathogenic fungi (Nectriaceae), insect-pathogenic fungi (Cordycipitaceae/Clavicipitaceae) and nematophagous fungi (Ophiocordycipitaceae). In addition, Subtilase genes from the nematode-parasitic fungus Purpureocillium lilacinum made a unique cluster which putatively indicated that the fungus might have developed distinctive mechanisms for nematode-pathogenesis. Our evolutionary genetics analysis revealed evidence of positive selection on the Subtilisin (S08.005) and Proteinase K (S08.054) protein sequences of the entomopathogenic and nematophagous species belonging to Cordycipitaceae, Clavicipitaceae and Ophiocordycipitaceae families of Hypocreales. CONCLUSIONS: Our study provided new insights into the evolution of Subtilisin like serine proteases in Hypocreales, a fungal order largely consisting of biological control species. Subtilisin (S08.005) and Proteinase K (S08.054) proteins seemed to play important roles during life style modifications among different families and species of Hypocreales. Protein residues found significant in functional divergence analysis in the present study may provide support for protein engineering in future.

Whole genome annotation and comparative genomic analyses of bio-control fungus Purpureocillium lilacinum.

BACKGROUND: The fungus Purpureocillium lilacinum is widely known as a biological control agent against plant parasitic nematodes. This research article consists of genomic annotation of the first draft of whole genome sequence of P. lilacinum. The study aims to decipher the putative genetic components of the fungus involved in nematode pathogenesis by performing comparative genomic analysis with nine closely related fungal species in Hypocreales. RESULTS: de novo genomic assembly was done and a total of 301 scaffolds were constructed for P. lilacinum genomic DNA. By employing structural genome prediction models, 13, 266 genes coding for proteins were predicted in the genome. Approximately 73% of the predicted genes were functionally annotated using Blastp, InterProScan and Gene Ontology. A 14.7% fraction of the predicted genes shared significant homology with genes in the Pathogen Host Interactions (PHI) database. The phylogenomic analysis carried out using maximum likelihood RAxML algorithm provided insight into the evolutionary relationship of P. lilacinum. In congruence with other closely related species in the Hypocreales namely, Metarhizium spp., Pochonia chlamydosporia, Cordyceps militaris, Trichoderma reesei and Fusarium spp., P. lilacinum has large gene sets coding for G-protein coupled receptors (GPCRs), proteases, glycoside hydrolases and carbohydrate esterases that are required for degradation of nematode-egg shell components. Screening of the genome by Antibiotics & Secondary Metabolite Analysis Shell (AntiSMASH) pipeline indicated that the genome potentially codes for a variety of secondary metabolites, possibly required for adaptation to heterogeneous lifestyles reported for P. lilacinum. Significant up-regulation of subtilisin-like serine protease genes in presence of nematode eggs in quantitative real-time analyses suggested potential role of serine proteases in nematode pathogenesis. CONCLUSIONS: The data offer a better understanding of Purpureocillium lilacinum genome and will enhance our understanding on the molecular mechanism involved in nematophagy.

Genome-wide association scan in north Indians reveals three novel HLA-independent risk loci for ulcerative colitis.

OBJECTIVE: Over 100 ulcerative colitis (UC) loci have been identified by genome-wide association studies (GWASs) primarily in Caucasians (CEUs). Many of them have weak effects on disease susceptibility, and the bulk of the heritability cannot be ascribed to these loci. Very little is known about the genetic background of UC in non-CEU groups. Here we report the first GWAS on UC in a genetically distinct north Indian (NI) population. DESIGN: A genome-wide scan was performed on 700 cases and 761 controls. 18 single-nucleotide polymorphisms (SNPs) (p<5×10(-5)) were genotyped in an independent cohort of 733 cases and 1148 controls. A linear mixed model was used for case-control association tests. RESULTS: Seven novel human leucocyte antigen (HLA)-independent SNPs from chromosome 6, located in 3.8-1, BAT2, MSH5, HSPA1L, SLC44A4, CFB and NOTCH4, exceeded p<5×10(-8) in the combined analysis. To assess the independent biological contribution of such genes from the extended HLA region, we determined the percentage alternative pathway activity of complement factor B (CFB), the top novel hit. The activity was significantly different (p=0.01) between the different genotypes at rs12614 in UC cases. Transethnic comparisons revealed a shared contribution of a fraction of UC risk genes between NI and CEU populations, in addition to genetic heterogeneity. CONCLUSIONS: This study shows varying contribution of the HLA region to UC in different populations. Different environmental exposures and the characteristic genetic structure of the HLA locus across ethnic groups collectively make it amenable to the discovery of causative alleles by transethnic resequencing. This may lead to an improved understanding of the molecular mechanisms underlying UC.

Case-control association analysis of dopamine receptor polymorphisms in alcohol dependence: a pilot study in Indian males.

BACKGROUND: Brain imaging studies and knock-out animal models have derived substantial abetment for dopamine receptor (DR) subtypes as potential candidates in susceptibility to addictive disorders, including alcohol dependence (AD). Various association studies that compared the frequencies of alleles of the dopamine D1, D2, D3 and D4 receptor genes between alcohol dependent and control subjects have produced suggestive results, though some of them are discordant in nature. In the absence of genetic data from Indian population, we evaluated genetic association of three polymorphisms namely rs4532 in DRD1, rs6280 in DRD3 and 120 bp duplication in 1.2 kb upstream region of DRD4 with AD. METHODS: A total of 90 cases (alcohol dependent males) and 122 age and ethnicity matched healthy male controls were recruited in the study by following DSM-IV criteria. Three polymorphisms, namely rs4532 in DRD1, rs6280 in DRD3 and 120 bp duplication in 1.2 kb upstream region of DRD4 were selected (based on minor allele frequency and available literature) for genotyping by PCR-RFLP/LP method. Allele and genotype frequencies of these genetic markers were compared using Pearson's χ2 test followed by risk assessment using odds ratio. Statistical analysis of clinical parameters such as AUDIT scores of case subjects was also performed. RESULTS: Statistically significant associations of polymorphisms in DRD1 and DRD4 with alcoholism were found. CONCLUSIONS: Our results underscore that genetic variations in dopamine receptors D1 and D4 may influence genetic predisposition to alcoholism. Unavailability of comparative data from Indian population and small sample size necessitate replication of results in an independent cohort.

A genome-wide association study reveals ARL15, a novel non-HLA susceptibility gene for rheumatoid arthritis in North Indians.

OBJECTIVE: Genome-wide association studies (GWAS) and their subsequent meta-analyses have changed the landscape of genetics in rheumatoid arthritis (RA) by uncovering several novel genes. Such studies are heavily weighted by samples from Caucasian populations, but they explain only a small proportion of total heritability. Our previous studies in genetically distinct North Indian RA cohorts have demonstrated apparent allelic/genetic heterogeneity between North Indian and Western populations, warranting GWAS in non-European populations. We undertook this study to detect additional disease-associated loci that may be collectively important in the presence or absence of genes with a major effect. METHODS: High-quality genotypes for >600,000 single-nucleotide polymorphisms (SNPs) in 706 RA patients and 761 controls from North India were generated in the discovery stage. Twelve SNPs showing suggestive association (P < 5 × 10(-5)) were then tested in an independent cohort of 927 RA patients and 1,148 controls. Additional disease-associated loci were determined using support vector machine (SVM) analyses. Fine-mapping of novel loci was performed by using imputation. RESULTS: In addition to the expected association of the HLA locus with RA, we identified association with a novel intronic SNP of ARL15 (rs255758) on chromosome 5 (Pcombined = 6.57 × 10(-6); odds ratio 1.42). Genotype-phenotype correlation by assaying adiponectin levels demonstrated the functional significance of this novel gene in disease pathogenesis. SVM analysis confirmed this association along with that of a few more replication stage genes. CONCLUSION: In this first GWAS of RA among North Indians, ARL15 emerged as a novel genetic risk factor in addition to the classic HLA locus, which suggests that population-specific genetic loci as well as those shared between Asian and European populations contribute to RA etiology. Furthermore, our study reveals the potential of machine learning methods in unraveling gene-gene interactions using GWAS data.

Caucasian and Asian specific rheumatoid arthritis risk loci reveal limited replication and apparent allelic heterogeneity in north Indians.

Genome-wide association studies and meta-analysis indicate that several genes/loci are consistently associated with rheumatoid arthritis (RA) in European and Asian populations. To evaluate the transferability status of these findings to an ethnically diverse north Indian population, we performed a replication analysis. We investigated the association of 47 single-nucleotide polymorphisms (SNPs) at 43 of these genes/loci with RA in a north Indian cohort comprising 983 RA cases and 1007 age and gender matched controls. Genotyping was done using Infinium human 660w-quad. Association analysis by chi-square test implemented in plink was carried out in two steps. Firstly, association of the index or surrogate SNP (r2>0.8, calculated from reference GIH Hap-Map population) was tested. In the second step, evidence for allelic/locus heterogeneity at aforementioned genes/loci was assessed for by testing additional flanking SNPs in linkage equilibrium with index/surrogate marker.Of the 44 European specific index SNPs, neither index nor surrogate SNPs were present for nine SNPs in the genotyping array. Of the remaining 35, associations were replicated at seven genes namely PTPN22 (rs1217407, p = 3×10(-3)); IL2-21 (rs13119723, p = 0.008); HLA-DRB1 (rs660895, p = 2.56×10(-5); rs6457617, p = 1.6×10(-09); rs13192471, p = 6.7×10(-16)); TNFA1P3 (rs9321637, p = 0.03); CCL21 (rs13293020, p = 0.01); IL2RA (rs2104286, p = 1.9×10(-4)) and ZEB1 (rs2793108, p = 0.006). Of the three Asian specific loci tested, rs2977227 in PADI4 showed modest association (p<0.02). Further, of the 140 SNPs (in LE with index/surrogate variant) tested, association was observed at 11 additional genes: PTPRC, AFF3, CD28, CTLA4, PXK, ANKRD55, TAGAP, CCR6, BLK, CD40 and IL2RB. This study indicates limited replication of European and Asian index SNPs and apparent allelic heterogeneity in RA etiology among north Indians warranting independent GWAS in this population. However, replicated associations of HLA-DRB1, PTPN22 (which confer ∼50% of the heritable risk to RA) and IL2RA suggest that cross-ethnicity fine mapping of such loci is apposite for identification of causal variants.

An investigation of genome-wide studies reported susceptibility loci for ulcerative colitis shows limited replication in north Indians.

Genome-Wide Association studies (GWAS) of both Crohn's Disease (CD) and Ulcerative Colitis (UC) have unearthed over 40 risk conferring variants. Recently, a meta-analysis on UC revealed several loci, most of which were either previously associated with UC or CD susceptibility in populations of European origin. In this study, we attempted to replicate these findings in an ethnically distinct north Indian UC cohort. 648 UC cases and 850 controls were genotyped using Infinium Human 660W-quad. Out of 59 meta-analysis index SNPs, six were not in the SNP array used in the study. Of the remaining 53 SNPs, four were found monomorphic. Association (p<0.05) at 25 SNPs was observed, of which 15 were CD specific. Only five SNPs namely rs2395185 (HLA-DRA), rs3024505 (IL10), rs6426833 (RNF186), rs3763313 (BTNL2) and rs2066843 (NOD2) retained significance after Bonferroni correction. These results (i) reveal limited replication of Caucasian based meta-analysis results; (ii) reiterate overlapping molecular mechanism(s) in UC and CD; (iii) indicate differences in genetic architecture between populations; and (iv) suggest that resources such as HapMap need to be extended to cover diverse ethnic populations. They also suggest a systematic GWAS in this terrain may be insightful for identifying population specific IBD risk conferring loci and thus enable cross-ethnicity fine mapping of disease loci.

Association analysis of ADPRT1, AKR1B1, RAGE, GFPT2 and PAI-1 gene polymorphisms with chronic renal insufficiency among Asian Indians with type-2 diabetes.

BACKGROUND: To determine association of nine single nucleotide polymorphisms (SNPs) in ADP ribosyltransferase-1 (ADPRT1), aldo-keto reductase family 1 member B1 (AKR1B1), receptor for advanced glycation end-products (RAGE), glutamine:fructose-6-phosphate amidotransferase-2 (GFPT2), and plasminogen activator inhibitor-1 (PAI-1) genes with chronic renal insufficiency (CRI) among Asian Indians with type 2 diabetes; and to identify epistatic interactionss between genes from the present study and those from renin-angiotensin-aldosterone system (RAAS), and chemokine-cytokine, dopaminergic and oxidative stress pathways (previously investigated using the same sample set). METHODS: Type 2 diabetes subjects with CRI (serum creatinine > or =3.0 mg/dl) constituted the cases (n = 196), and ethnicity and age matched individuals with diabetes for a duration of > or = 10 years, normal renal functions and normoalbuminuria recruited as controls (n = 225). Allelic and genotypic constitution of 10 polymorphisms (SNPs) from five genes namely--ADPRT1, AKR1B1, RAGE, GFPT2 and PAI-1 with diabetic CRI was investigated. The genetic associations were evaluated by computation of odds ratio and 95% confidence interval. Multiple logistic regression analysis was carried out to correlate various clinical parameters with genotypes, and to study epistatic interactions between SNPs in different genes. RESULTS: Single nucleotide polymorphisms -429 T>C in RAGE and rs7725 C>T SNP in 3' UTR in GFPT2 gene showed a trend towards association with diabetic CRI. Investigation using miRBase statistical tool revealed that rs7725 in GFPT2 was a perfect target for predicted miRNA (hsa miR-378) suggesting the presence of the variant 'T' allele may result in an upregulation of GFPT2 contributing to diabetic renal complication. Epistatic interaction between SNPs in transforming growth factor TGF-beta1 (investigated using the same sample set and reported elsewhere) and GFPT2 genotype was observed. CONCLUSIONS: Association of SNPs in RAGE and GFPT2 suggest that the genes involved in modulation of oxidative pathway could be major contributor to diabetic chronic renal insufficiency. In addition, GFPT2 mediated overproduction of TGF-beta1 leading to endothelial expansion and thereby CRI seems likely, suggested by our observation of a significant interaction between GFPT2 with TGF-beta1 genes. Further, identification of predicted miRNA targets spanning the associated SNP in GFPT2 implicates the rs7725 SNP in transcriptional regulation of the gene, and suggests GFPT2 could be a relevant target for pharmacological intervention. Larger replication studies are needed to confirm these observations.

Dopamine D2 receptor polymorphisms and susceptibility to alcohol dependence in Indian males: a preliminary study.

BACKGROUND: Dopamine is an important neurotransmitter involved in reward mechanism in the brain and thereby influences development and relapse of alcohol dependence. The dopamine D2 receptor (DRD2) gene on chromosome 11 (q22-q23) has been found to be associated with increased alcohol consumption through mechanisms involving incentive salience attributions and craving in alcoholic patients. Therefore, we investigated the association of three single nucleotide polymorphisms (SNP) in DRD2 gene with alcohol dependence in the north Indian subjects. METHODS: In a retrospective analysis, genetic association of three polymorphisms from DRD2 gene with alcohol dependence was investigated using a case-control approach. Alcohol dependence was determined by DSM-IV criteria and a total of 90 alcoholics and 60 healthy unrelated age-matched control subjects were recruited. Odds ratio and confidence interval was calculated to determine risk conferred by a predisposing allele/genotype/haplotype. Logistic regression analysis was carried out to correlate various clinical parameters with genotypes, and to study pair-wise interactions between SNPs. RESULTS: The study showed a significant association of -141C Ins allele and a trend of association of TaqI A1 allele of DRD2 with alcohol dependence. Haplotype with the predisposing -141C Ins and TaqI A1 alleles (-141C Ins-A-A1) seems to confer approximately 2.5 times more risk to develop alcohol dependence. CONCLUSIONS: The study provides preliminary insight into genetic risk to alcohol dependence in Indian males. Two polymorphisms namely, -141C Ins/Del and TaqI A in DRD2 gene may have clinical implications among Indian alcoholic subjects.

Association of ADH1B and ALDH2 gene polymorphisms with alcohol dependence: a pilot study from India.

Functional polymorphism in the genes encoding alcohol dehydrogenase (ADH) 1B and aldehyde dehydrogenase (ALDH) 2 are considered most important among several genetic determinants of alcohol dependence, a complex disorder. There is no report on the widely studied Arg47His and Glu487Lys polymorphisms from Indian alcohol-dependent populations. In this paper, we report, for the first time, allelic and genotypic frequencies of Arg47His and Glu487Lys single nucleotide polymorphisms (SNPs) in North Indian alcohol-dependent subjects. A total of 174 alcohol-dependent males, recruited using DSM IV criteria (American Psychiatric Association, 1994), were genotyped using the polymerase chain reaction-restriction fragment length polymorphism method. The results obtained from genetic analysis were correlated with clinical parameters using Student's t-test or Mann Whitney's U test. The highlight of the study findings was the uniquely high frequency of the ALDH2*2/*2 genotype (among alcohol-dependent subjects) being a risk-conferring factor for alcohol dependence.

Oxidative stress pathway genes and chronic renal insufficiency in Asian Indians with Type 2 diabetes.

BACKGROUND: There are significant regional variations in prevalence of diabetes and diabetic chronic renal insufficiency (CRI) in India. Oxidative stress plays an important role in the development of diabetic complications. To determine the importance of the polymorphisms in the genes involved in maintenance of cellular redox balance, we performed a case control study in subjects from south and north India. METHODS: Successive cases presenting to the study centers with Type 2 diabetes of >2 years duration and moderate CRI (n=194, south India 104, north India 90) diagnosed by serum creatinine >or=2 mg/dl after exclusion of nondiabetic causes of CRI were compared with diabetes subjects with no evidence of renal disease (n=224, south India 149, north India 75). Twenty-six polymorphisms from 13 genes from the oxidative stress pathway were analyzed using polymerase chain reaction-restriction fragment length polymorphism. Genes included were superoxide dismutases (SOD1, 2, 3), uncoupling proteins (UCP1, 2), endothelial nitric oxide synthase (NOS3), glutathione-S-transferases (GST) (M1, T1, P1), vascular endothelial growth factor (VEGF), paraoxonase (PON) 1 and 2, and nicotinamide adenine dinucleotide phosphate reduced, oxidase p22(phox). Genes were tested for their association with CRI using chi(2) test. RESULTS: In south Indian (SI) subjects there was significant allelic and genotypic association of the wild-type allele in SOD2 (Ala9Val; P=.002 and P=.013, respectively), UCP1 (-112 T>G, P=.012 and P=.009; Ala64Thr, P=.015 and P=.004), NOS3 (Glu298Asp, P=.002 and P=.009) and GSTP1 (Ile105Val, P=.003 and P=.004) genes with development of CRI. None of these observations were replicated in the north Indian (NI) subjects. A genotypic but not allelic association was observed for two markers, VEGF (-460 T>C) and PON1 (Arg192Gly) among NI diabetic CRI subjects. CONCLUSION: The nonreplication of association suggests differential genetic susceptibility of the two populations to diabetic chronic renal insufficiency. In the SI diabetic subjects, oxidative stress pathway genes might be an important predictor for the development of diabetic complications. Further, the association of wild-type alleles may suggest that they confer greater survival ability to comorbid complications and may be nephroprotective.

Association of dopaminergic pathway gene polymorphisms with chronic renal insufficiency among Asian Indians with type-2 diabetes.

BACKGROUND: Genetic markers conferring susceptibility to diabetes specific renal disease remains to be identified for early prediction and development of effective drugs and therapies. Inconsistent results obtained from analysis of genes from classical pathways generate need for examination of unconventional genetic markers having role in regulation of renal function. Experimental and clinical evidences suggest that dopamine is an important natriuretic hormone. Therefore, various genes involved in regulation of dopamine bioavailability could play a role in diabetic chronic renal insufficiency (CRI). We investigated the contribution of 12 polymorphisms from five Dopaminergic pathway genes to CRI among type-2 diabetic Asian Indian subjects. METHODS: Genetic association of 12 polymorphisms (SNPs) from five genes namely-dopamine receptor-1 (DRD1), DRD2, DRD3, DRD4, andcatechol-O-methyltransferase (COMT) with diabetic CRI was investigated using a case-control approach. Logistic regression analysis was carried out to correlate various clinical parameters with genotypes, and to study pair wise interactions between SNPs of different genes. RESULTS: SNPs -141 ins/del C and G>A (1 kb upstream from exon 2) in DRD2 gene showed significant allelic and genotypic association. Allele -141 insC and genotype -141 insC/insC of -141 ins/del C polymorphism, and allele A of G>A SNP were found to be predisposing to CRI. Our result of allelic and genotypic association of -141 insC/delC SNP was also reflected in the haplotypic association. Heterozygous genotype of polymorphism 900 ins/del C in COMT gene was predisposing towards CRI. CONCLUSION: Some polymorphisms in DRD2 and COMT genes are significantly associated with susceptibility to CRI in the Asian Indian population which, if confirmed would be consistent with a suggested role of dopamine metabolism in disease occurrence.

Normative genetic profiles of RAAS pathway gene polymorphisms in North Indian and South Indian populations.

Population-based genetic association studies, popularly known as case-control studies, have continued to be the most preferred method for deciphering the genetic basis of various complex diseases, even in the post-human genome sequencing era. However, interpopulation differences in allele, genotype, and haplotype frequencies and linkage disequilibrium patterns lead to inconsistent results in candidate gene association studies. Therefore, for any meaningful disease association study, knowledge of the normative genetic background of the baseline population is a prerequisite. In addition, such genetic variation data also provide a ready-made menu of allele frequencies and linkage disequilibrium patterns of various polymorphisms in specific candidate genes in a particular population, which is a useful reference for further genetic association studies. Such genetic variation data are lacking for the Indian population, which represents about one-sixth of the world's population. In the present study we have reported the allele, genotype, and haplotype frequencies, Hardy-Weinberg equilibrium status, and linkage disequilibrium patterns of 12 polymorphisms in six candidate genes from the renin-angiotensin-aldosterone system among Indians. Because of their different history of origin, the Indian population is broadly divided into two subpopulations: North Indians (Caucasian Europeans) and South Indians (Dravidians). Considering this well-documented difference in gene pools, we have presented a comparative account of the normative genetic data of North Indian and South Indian populations with at least four individuals of urban and suburban origin from each of the representative states of northern and southern India.

Association of TGFbeta1, TNFalpha, CCR2 and CCR5 gene polymorphisms in type-2 diabetes and renal insufficiency among Asian Indians.

BACKGROUND: Cytokines play an important role in the development of diabetic chronic renal insufficiency (CRI). Transforming growth factor beta1 (TGF beta1) induces renal hypertrophy and fibrosis, and cytokines like tumor necrosis factor-alpha (TNFalpha), chemoattractant protein-1 (MCP-1), and regulated upon activation and normal T cell expressed and secreted (RANTES) mediate macrophage infiltration into kidney. Over expression of these chemokines leads to glomerulosclerosis and interstitial fibrosis. The effect of MCP-1 and RANTES on kidney is conferred by their receptors i.e., chemokine receptor (CCR)-2 and CCR-5 respectively. We tested association of nine single nucleotide polymorphisms (SNPs) from TGFbeta1, TNFalpha, CCR2 and CCR5 genes among individuals with type-2 diabetes with and without renal insufficiency. METHODS: Type-2 diabetes subjects with chronic renal insufficiency (serum creatinine > or = 3.0 mg/dl) constituted the cases, and matched individuals with diabetes of duration > or = 10 years and normoalbuminuria were evaluated as controls from four centres in India. Allelic and genotypic contributions of nine SNPs from TGFbeta1, TNFalpha, CCR2 and CCR5 genes to diabetic CRI were tested by computing odds ratio (OR) and 95% confidence intervals (CI). Sub-analysis of CRI cases diabetic retinopathy status as dependent variable and SNP genotypes as independent variable in a univariate logistic regression was also performed. RESULTS: SNPs Tyr81His and Thr263Ile in TGF beta1 gene were monomorphic, and Arg25Pro in TGF beta1 gene and Delta32 polymorphism in CCR5 gene were minor variants (minor allele frequency <0.05) and therefore were not considered for case-control analysis. A significant allelic association of 59029G>A SNP of CCR5 gene has been observed and the allele 59029A seems to confer predisposition to development of diabetic CRI (OR 1.39; CI 1.04-1.84). In CRI subjects a compound group of genotypes "GA and AA" of SNP G>A -800 was found to confer predisposition for proliferative retinopathy (OR 3.03; CI 1.08-8.50, p = 0.035). CONCLUSION: Of the various cytokine gene polymorphisms tested, allele 59029A of CCR5 gene is significantly associated with diabetic renal insufficiency among Asian Indians. Result obtained for 59029G>A SNP of CCR5 gene is in conformity with reports from a Japanese population but due to sub-optimal power of the sample, replication in larger sample set is warranted.

Chronic renal insufficiency among Asian Indians with type 2 diabetes: I. Role of RAAS gene polymorphisms.

BACKGROUND: Renal failure in diabetes is mediated by multiple pathways. Experimental and clinical evidences suggest that renin-angiotensin-aldosterone system (RAAS) has a crucial role in diabetic kidney disease. A relationship between the RAAS genotypes and chronic renal insufficiency (CRI) among type 2 diabetes subjects has therefore been speculated. We investigated the contribution of selected RAAS gene polymorphisms to CRI among type 2 diabetic Asian Indian subjects. METHODS: Twelve single nucleotide polymorphisms (SNPs) from six genes namely-renin (REN), angiotensinogen (ATG), angiotensin converting enzyme I (ACE), angiotensin II type 1 receptor (AT1) and aldosterone synthase (CYP11B2) gene from the RAAS pathway and one from chymase pathway were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and tested for their association with diabetic CRI using a case-control approach. Successive cases presenting to study centres with type 2 diabetes of > or =2 years duration and moderate CRI diagnosed by serum creatinine > or =3 mg/dl after exclusion of non-diabetic causes of CRI (n = 196) were compared with diabetes subjects with no evidence of renal disease (n = 225). Logistic regression analysis was carried out to correlate various clinical parameters with genotypes, and to study pair wise interactions between SNPs of different genes. RESULTS: Of the 12 SNPs genotyped, Glu53Stop in AGT and A>T (-777) in AT1 genes, were monomorphic and not included for further analysis. We observed a highly significant association of Met235Thr SNP in angiotensinogen gene with CRI (O.R. 2.68, 95%CI: 2.01-3.57 for Thr allele, O.R. 2.94, 95%CI: 1.88-4.59 for Thr/Thr genotype and O.R. 2.68, 95%CI: 1.97-3.64 for ACC haplotype). A significant allelic and genotypic association of T>C (-344) SNP in aldosterone synthase gene (O.R. 1.57, 95%CI: 1.16-2.14 and O.R. 1.81, 95%CI: 1.21-2.71 respectively), and genotypic association of GA genotype of G>A (-1903) in chymase gene (O.R. 2.06, 95%CI: 1.34-3.17) were also observed. CONCLUSION: SNPs Met235Thr in angiotensinogen, T>C (-344) in aldosterone synthase, and G>A (-1903) in chymase genes are significantly associated with diabetic chronic renal insufficiency in Indian patients and warrant replication in larger sample sets. Use of such markers for prediction of susceptibility to diabetes specific renal disease in the ethnically Indian population appears promising.