Saman Habib

The Indian Genome Variation database (IGVdb): A project overview

Indian population, comprising of more than a billion people, consists of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts. To address the questions related to ethnic diversity, migrations, founder populations, predisposition to complex disorders or pharmacogenomics, one needs to understand the diversity and relatedness at the genetic level in such a diverse population. In this backdrop, six constituent laboratories of the Council of Scientific and Industrial Research (CSIR), with funding from the Government of India, initiated a network program on predictive medicine using repeats and single nucleotide polymorphisms. The Indian Genome Variation (IGV) consortium aims to provide data on validated SNPs and repeats, both novel and reported, along with gene duplications, in over a thousand genes, in 15,000 individuals drawn from Indian subpopulations. These genes have been selected on the basis of their relevance as functional and positional candidates in many common diseases including genes relevant to pharmacogenomics. This is the first large-scale comprehensive study of the structure of the Indian population with wide-reaching implications. A comprehensive platform for Indian Genome Variation (IGV) data management, analysis and creation of IGVdb portal has also been developed. The samples are being collected following ethical guidelines of Indian Council of Medical Research (ICMR) and Department of Biotechnology (DBT), India. This paper reveals the structure of the IGV project highlighting its various aspects like genesis, objectives, strategies for selection of genes, identification of the Indian subpopulations, collection of samples and discovery and validation of genetic markers, data analysis and monitoring as well as the project’s data release policy.Indian population, comprising of more than a billion people, consists of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts. To address the questions related to ethnic diversity, migrations, founder populations, predisposition to complex disorders or pharmacogenomics, one needs to understand the diversity and relatedness at the genetic level in such a diverse population. In this backdrop, six constituent laboratories of the Council of Scientific and Industrial Research (CSIR), with funding from the Government of India, initiated a network program on predictive medicine using repeats and single nucleotide polymorphisms. The Indian Genome Variation (IGV) consortium aims to provide data on validated SNPs and repeats, both novel and reported, along with gene duplications, in over a thousand genes, in 15,000 individuals drawn from Indian subpopulations. These genes have been selected on the basis of their relevance as functional and positional candidates in many common diseases including genes relevant to pharmacogenomics. This is the first large-scale comprehensive study of the structure of the Indian population with wide-reaching implications. A comprehensive platform for Indian Genome Variation (IGV) data management, analysis and creation of IGVdb portal has also been developed. The samples are being collected following ethical guidelines of Indian Council of Medical Research (ICMR) and Department of Biotechnology (DBT), India. This paper reveals the structure of the IGV project highlighting its various aspects like genesis, objectives, strategies for selection of genes, identification of the Indian subpopulations, collection of samples and discovery and validation of genetic markers, data analysis and monitoring as well as the project’s data release policy.

Polymorphisms of TNF-enhancer and gene for FcγRIIa correlate with the severity of falciparum malaria in the ethnically diverse Indian population

BackgroundSusceptibility/resistance to Plasmodium falciparum malaria has been correlated with polymorphisms in more than 30 human genes with most association analyses having been carried out on patients from Africa and south-east Asia. The aim of this study was to examine the possible contribution of genetic variants in the TNF and FCGR2A genes in determining severity/resistance to P. falciparum malaria in Indian subjects.MethodsAllelic frequency distribution in populations across India was first determined by typing genetic variants of the TNF enhancer and the FCGR2A G/A SNP in 1871 individuals from 55 populations. Genotyping was carried out by DNA sequencing, single base extension (SNaPshot), and DNA mass array (Sequenom). Plasma TNF was determined by ELISA. Comparison of datasets was carried out by Kruskal-Wallis and Mann-Whitney tests. Haplotypes and LD plots were generated by PHASE and Haploview, respectively. Odds ratio (OR) for risk assessment was calculated using EpiInfo™ version 3.4.ResultsA novel single nucleotide polymorphism (SNP) at position -76 was identified in the TNF enhancer along with other reported variants. Five TNF enhancer SNPs and the FCGR2A R131H (G/A) SNP were analyzed for association with severity of P. falciparum malaria in a malaria-endemic and a non-endemic region of India in a case-control study with ethnically-matched controls enrolled from both regions. TNF -1031C and -863A alleles as well as homozygotes for the TNF enhancer haplotype CACGG (-1031T>C, -863C>A, -857C>T, -308G>A, -238G>A) correlated with enhanced plasma TNF levels in both patients and controls. Significantly higher TNF levels were observed in patients with severe malaria. Minor alleles of -1031 and -863 SNPs were associated with increased susceptibility to severe malaria. The high-affinity IgG2 binding FcγRIIa AA (131H) genotype was significantly associated with protection from disease manifestation, with stronger association observed in the malaria non-endemic region. These results represent the first genetic analysis of the two immune regulatory molecules in the context of P. falciparum severity/resistance in the Indian population.ConclusionAssociation of specific TNF and FCGR2A SNPs with cytokine levels and disease severity/resistance was indicated in patients from areas with differential disease endemicity. The data emphasizes the need for addressing the contribution of human genetic factors in malaria in the context of disease epidemiology and population genetic substructure within India.

The apicoplast of Plasmodium falciparum is translationally active

Apicoplast, the plastid‐like organelle of apicomplexan parasites, has generated interest as a putative drug target. Although transcripts for genes encoded by the 35 kb circular plastid DNA have been detected, the actual presence of their protein products has only been postulated. We provide evidence for translation of the tufA gene encoded by the Plasmodium falciparum apicoplast genome. Translation elongation factor Tu (EF‐Tu), the product of tufA, was localized within the organelle. TufA was found to express maximally in the trophozoite stage of the intraerythrocytic cycle. Additionally, the drug thiostrepton that has a binding site in apicoplast LSU rRNA, reduced P. falciparum apicoplast EF‐Tu levels thus strengthening the view that translation in the apicoplast is the site of action of this drug.

Protein translation in Plasmodium parasites.

The protein translation machinery of the parasite Plasmodium is the target of important anti-malarial drugs, and encompasses many promising targets for future drugs. Plasmodium parasites have three subcellular compartments that house genomes; the nucleus, mitochondrion and apicoplast, and each requires its own compartmentalized transcription and translation apparatus for survival. Despite the availability of the complete genome sequence that should reveal the requisite elements for all three compartments, our understanding of the translation machineries is patchy. We review what is known about cytosolic and organellar translation in Plasmodium and discuss the molecules that have been identified through genome sequencing and post-genomic analysis. Some translation components are yet to be found in Plasmodium, whereas others appear to be shared between translationally active organelles.

Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies

Nitric oxide (NO) modulates diverse functions of polymorphonuclear neutrophils (PMNs), but localization of NO synthase (NOS) and identification of its interacting proteins remain the least defined. The present study discerns subcellular distribution of NOS and caveolin‐1, a prominent NOS‐interacting protein in rat PMNs. Localization of NOS was explored by confocal and immunogold electron microscopy, and its activity was assessed by L‐[3H] arginine and 4,5‐diaminofluorescein diacetate (DAF‐2DA). Reverse transcriptase‐polymerase chain reaction using NOS primers and Western blotting demonstrated the presence of neuronal NOS (nNOS) and inducible NOS (iNOS) in PMNs. Immunocytochemical studies exhibited distribution of nNOS and iNOS in cytoplasm and nucleus, and L‐[3H] citrulline formation and DAF fluorescence confirmed NOS activity in both fractions. NOS activity correlated positively with calmodulin concentration in both of the fractions. nNOS and iNOS colocalized with caveolin‐1, as evidenced by immunocytochemical and immunoprecipitation studies. The results thus provide first evidence of nNOS and iNOS in the nuclear compartment and suggest NOS interaction with caveolin‐1 in rat PMNs.

DNA organization by the apicoplast-targeted bacterial histone-like protein of Plasmodium falciparum

Apicomplexans, including the pathogens Plasmodium and Toxoplasma, carry a nonphotosynthetic plastid of secondary endosymbiotic origin called the apicoplast. The P. falciparum apicoplast contains a 35 kb, circular DNA genome with limited coding capacity that lacks genes encoding proteins for DNA organization and replication. We report identification of a nuclear-encoded bacterial histone-like protein (PfHU) involved in DNA compaction in the apicoplast. PfHU is associated with apicoplast DNA and is expressed throughout the parasites intra-erythocytic cycle. The protein binds DNA in a sequence nonspecific manner with a minimum binding site length of ∼27 bp and a Kd of ∼63 nM and displays a preference for supercoiled DNA. PfHU is capable of condensing Escherichia coli nucleoids in vivo indicating its role in DNA compaction. The unique 42 aa C-terminal extension of PfHU influences its DNA condensation properties. In contrast to bacterial HUs that bend DNA, PfHU promotes concatenation of linear DNA and inhibits DNA circularization. Atomic Force Microscopic study of PfHU–DNA complexes shows protein concentration-dependent DNA stiffening, intermolecular bundling and formation of DNA bridges followed by assembly of condensed DNA networks. Our results provide the first functional characterization of an apicomplexan HU protein and provide additional evidence for red algal ancestry of the apicoplast.

Differential Activity of Two Non-hr Origins during Replication of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus Genome

ABSTRACT The identification of potential baculovirus origins of replication (ori) has involved the generation and characterization of defective interfering particles that contain major genomic deletions yet retain their capability to replicate by testing the replication ability of transiently transfected plasmids carrying viral sequences in infected cells. So far, there has not been any evidence to demonstrate the actual utilization of these putative origins in Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) replication. By using the method of origin mapping by competitive PCR, we have obtained quantitative data for theori activity of the HindIII-K region and theie-1 promoter sequence in AcMNPV. We also provide evidence for differential activity of the two oriin the context of the viral genome through the replication phase of viral infection. Comparison of the number of molecules representing theHindIII-K and ie-1 origins vis-à-vis the non-ori polH region in a size-selected nascent DNA preparation revealed that the HindIII-K ori is utilized ∼14 times more efficiently than the ie-1 region during the late phase of infection. HindIII-K also remains the more active ori through the early and middle replication phases. Our results provide in vivo evidence in support of the view that AcMNPV replication involves multipleori that are activated with vastly different efficiencies during the viral infection cycle.

Interaction between sulphur mobilisation proteins SufB and SufC: Evidence for an iron-sulphur cluster biogenesis pathway in the apicoplast of Plasmodium falciparum

The plastid of Plasmodium falciparum, the apicoplast, performs metabolic functions essential to the parasite. Various reactions in the plastid require the assembly of [Fe-S] prosthetic groups on participating proteins as well as the reductant activity of ferredoxin that is converted from its apo-form by the assembly of [Fe-S] clusters inside the apicoplast. The [Fe-S] assembly pathway involving sulphur mobilising Suf proteins has been predicted to function in the apicoplast with one component (PfSufB) encoded by the plastid genome itself. We demonstrate the ATPase activity of recombinant P. falciparum nuclear-encoded SufC and its localisation in the apicoplast. Further, an internal region of apicoplast SufB was used to detect PfSufB-PfSufC interaction in vitro; co-elution of SufB from parasite lysate with recombinant PfSufC on an affinity column also indicated an interaction of the two proteins. As a departure from bacterial SufB and similar to reported plant plastid SufB, apicoplast SufB exhibited ATPase activity, suggesting the evolution of specialised functions in the plastid counterparts. Our results provide experimental evidence for an active Suf pathway in the Plasmodium apicoplast.

CR1 levels and gene polymorphisms exhibit differential association with falciparum malaria in regions of varying disease endemicity.

Complement receptor 1 (CR1/CD35) levels on erythrocytes and related CR1 polymorphisms have been associated with response to falciparum malaria in populations inhabiting malaria-endemic regions. Differences in disease association profiles of its low expression alleles have been observed in populations from different regions of the world. We analyzed the influence of CR1 levels and associated SNPs on susceptibility/resistance to falciparum malaria in Indian populations. Two CR1 SNPs [exon 22 (A/G) and intron 27 (A/T)] define the low expression (L) CR1 allele in populations inhabiting a Plasmodium falciparum-endemic and a nonendemic region of India. Populations of the endemic region have very low red blood cell surface CR1 levels and higher frequencies of the exon 22 and intron 27 mutant L alleles. Whereas low CR1 levels correlated with susceptibility to severe malaria in the nonendemic region, high CR1 levels were associated with manifestation of disease in the endemic region. In addition, the exon 22 L allele was a risk factor for severe malaria in the nonendemic region. Absence of correlation between levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 with CR1 levels in patients with severe disease indicated that RBC CR1 levels in individuals are not the major determinants of pro-inflammatory cytokine release during infection. Our results are interpreted in the context of differences in the pathogenesis of severe malaria in the malaria-endemic and nonendemic region.

Molecular and biochemical characterization of nitric oxide synthase isoforms and their intracellular distribution in human peripheral blood mononuclear cells.

Nitric oxide synthase (NOS) expression and catalytic status in human peripheral blood mononuclear cells (PBMCs) is debatable, while its sub-cellular distribution remains unascertained. The present study characterizes NOS transcripts by real time PCR, NOS protein by immunoprecipitation (IP)/Western blot (WB), nitric oxide (NO) generation by DAF-2DA and NOS sub-cellular distribution by immunogold electron microscopy in resting PBMCs, monocytes and lymphocytes obtained from healthy donors. We observed constitutive expression of full length NOS isoforms (nNOS, iNOS and eNOS) in PBMCs: with the highest expression of iNOS in comparison to nNOS and eNOS. Isolated monocytes expressed more eNOS transcript and protein as compared to nNOS and iNOS. Lymphocytes however had more iNOS transcripts and protein than nNOS and eNOS. NOS was catalytically active in PBMCs, monocytes as well as in lymphocytes as evident by NO generation in the presence of substrate and cofactors, which was significantly reduced in the presence of NOS inhibitor. Immunogold electron microscopy and morphometric analysis revealed the distinct pattern of NOS distribution in monocytes and lymphocytes and also exhibited differences in the nuclear-cytoplasmic ratio. nNOS localization was much more in the cytosol than in the nucleus among both monocytes and lymphocytes. Interestingly, iNOS distribution was comparable in both cytosol and nucleus among monocytes, but in lymphocytes iNOS was predominantly localized to the cytosol. The present study exhibits constitutive presence of all the NOS isoforms in PBMCs and reports the distinct pattern of NOS distribution among monocytes and lymphocytes.