Christopher S. Peacock

Comparative genomic analysis of three Leishmania species that cause diverse human disease.

Leishmania parasites cause a broad spectrum of clinical disease. Here we report the sequencing of the genomes of two species of Leishmania: Leishmania infantum and Leishmania braziliensis. The comparison of these sequences with the published genome of Leishmania major reveals marked conservation of synteny and identifies only ∼200 genes with a differential distribution between the three species. L. braziliensis, contrary to Leishmania species examined so far, possesses components of a putative RNA-mediated interference pathway, telomere-associated transposable elements and spliced leader–associated SLACS retrotransposons. We show that pseudogene formation and gene loss are the principal forces shaping the different genomes. Genes that are differentially distributed between the species encode proteins implicated in host-pathogen interactions and parasite survival in the macrophage.

GeneDB: a resource for prokaryotic and eukaryotic organisms

GeneDB (http://www.genedb.org/) is a genome database for prokaryotic and eukaryotic organisms. The resource provides a portal through which data generated by the Pathogen Sequencing Unit at the Wellcome Trust Sanger Institute and other collaborating sequencing centres can be made publicly available. It combines data from finished and ongoing genome and expressed sequence tag (EST) projects with curated annotation, that can be searched, sorted and downloaded, using a single web based resource. The current release stores 11 datasets of which six are curated and maintained by biologists, who review and incorporate information from the scientific literature, public databases and the respective research communities.

SLC11A1 (formerly NRAMP1) and disease resistance

Slc11a1 (formerly Nramp1) has many pleiotropic effects on macrophage (m) activation, including regulation of the CXC chemokine KC, interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), major histocompatibility complex (MHC) class II molecules, tumour necrosis factor α (TNFα), nitric oxide (NO) release, l-arginine flux, oxidative burst and tumoricidal as well as antimicrobial activity (reviewed by Blackwell and Searle, 1999; Blackwell et al., 2000). A naturally occurring Gly→Asp mutation at amino acid 169 of Slc11a1 makes mice as susceptible to Leishmania donovani, Salmonella typhimurium and Mycobacterium bovis as gene-disrupted mice (Vidal et al., 1995). Hence, the mutation is a functional null. This mutation also confers susceptibility to a range of other pathogens in mice, including Mycobacterium lepraemurium (Brown et al., 1982; Skamene et al., 1984), Mycobacterium intracellulare (Goto et al., 1989), Toxoplasma gondii (Blackwell et al., 1994), Candida albicans (Puliti et al., 1995) and Leishmania infantum (Leclercq et al., 1996). In man, SLC11A1 is linked or associated with multiple infectious (Shaw et al., 1997; Abel et al., 1998; Bellamy et al., 1998; Marquet et al., 1999; Cervino et al., 2000; Gao et al., 2000; Greenwood et al., 2000; Ryu et al., 2000; Mohamed et al., 2001) and autoimmune (Shaw et al., 1996; Hofmeister et al., 1997; Esposito et al., 1998; Maliarik et al., 2000; Sanjeevi et al., 2000; Singal et al., 2000; Yang et al., 2000) diseases. The infectious diseases include viral (HIV), bacterial (tuberculosis, leprosy, meningococcal meningitis) and protozoan (visceral leishmaniasis) pathogens. The autoimmune diseases include rheumatoid arthritis, juvenile rheumatoid arthritis, diabetes, sarcoidosis and Crohns disease. Mutation in the closely related Slc11a2 (Nramp2) gene causes microcytic anaemia in mice (Fleming et al., 1997), but disease association in man has not been reported. Slc11a1 and Slc11a2 are polytopic integral membrane proteins with 10–12 putative membrane-spanning domains (Vidal et al., 1993; Gunshin et al., 1997). In both, the natural functional null mutation occurs in transmembrane domain 4 (Vidal et al., 1993; Fleming et al., 1997). Both Slc11a1 and Slc11a2 have protein kinase C (PKC) binding sites (Vidal et al., 1993; Barton et al., 1994; Gruenheid et al., 1995), but only Slc11a1 has a Pro–Ser-rich N-terminus (Barton et al., 1994). Here, we review current knowledge on the evolution, function and roles of Slc11a1/SLC11A1 in disease.

Genetic and Epigenetic Factors at COL2A1 and ABCA4 Influence Clinical Outcome in Congenital Toxoplasmosis

Background Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute. Methods and Findings In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mothers genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting. Conclusions These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite.

Evidence for a cluster of genes on chromosome 17q11–q21 controlling susceptibility to tuberculosis and leprosy in Brazilians

The region of conserved synteny on mouse chromosome 11/human 17q11–q21 is known to carry a susceptibility gene(s) for intramacrophage pathogens. The region is rich in candidates including NOS2A, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCR7, STAT3 and STAT5A/5B. To examine the region in man, we studied 92 multicase tuberculosis (627 individuals) and 72 multicase leprosy (372 individuals) families from Brazil. Multipoint nonparametric analysis (ALLEGRO) using 16 microsatellites shows two peaks of linkage for leprosy at D17S250 (Zlr score 2.34; P=0.01) and D17S1795 (Zlr 2.67; P=0.004) and a single peak for tuberculosis at D17S250 (Zlr 2.04; P=0.02). Combined analysis shows significant linkage (peak Zlr 3.38) at D17S250, equivalent to an allele sharing LOD score 2.48 (P=0.0004). To determine whether one or multiple genes contribute, 49 informative single nucleotide polymorphisms were typed in candidate genes. Family-based allelic association testing that was robust to family clustering demonstrated significant associations with tuberculosis susceptibility at four loci separated by intervals (NOS2A–8.4 Mb–CCL18–32.3 kb–CCL4–6.04 Mb–STAT5B) up to several Mb. Stepwise conditional logistic regression analysis using a case/pseudo-control data set showed that the four genes contributed separate main effects, consistent with a cluster of susceptibility genes across 17q11.2.

Evidence that genetic susceptibility to Mycobacterium tuberculosis in a brazilian population is under oligogenic control: Linkage study of the candidate genes NRAMP1 and TBFA

SETTING A study of multicase tuberculosis pedigrees from Northern Brazil. OBJECTIVE To determine the model of inheritance for genetic susceptibility to tuberculosis, and to test the hypothesis that TNFA and NRAMP1 are candidate susceptibility genes. DESIGN The study sample included 98 pedigrees, 704 individuals and 205 nuclear families. Segregation analyses were performed using the programs POINTER and COMDS. Combined segregation and linkage analysis was carried out within COMDS. Non-parametric linkage analyses were performed using BETA. RESULTS A sporadic model for disease distribution in families was strongly rejected, as were polygenic and multifactorial models. A codominant single gene model provided the best fit (P < 0.001) to the data using POINTER. COMDS extended the analysis to compare single-gene and two-gene models. A general two-locus model for disease control was marginally favoured (0.01 < P < 0.05) over the codominant single-gene model. No evidence was found for linkage between susceptibility to disease per se and the TNF gene cluster. Weak linkage was observed using COMDS for genes (IL8RB, P = 0.039; D2S1471, P = 0.025) tightly linked (< 150 kb) to NRAMP1, but not for NRAMP1 itself. CONCLUSIONS Tuberculosis susceptibility in this region of Brazil is under oligogenic control. Although a minor role for TNFA and NRAMP1 cannot be excluded, our data suggest that neither is a major gene involved in this oligogenic control.

Genome-wide scans for leprosy and tuberculosis susceptibility genes in Brazilians

Genome-wide scans were conducted for tuberculosis and leprosy per se in Brazil. At stage 1, 405 markers (10 cM map) were typed in 16 (178 individuals) tuberculosis and 21 (173 individuals) leprosy families. Nonparametric multipoint analysis detected 8 and 9 chromosomal regions respectively with provisional evidence (P<0.05) for linkage. At stage 2, 58 markers from positive regions were typed in a second set of 22 (176 individuals) tuberculosis families, with 22 additional markers typed in all families; 42 positive markers in 50 (192 individuals) new leprosy families, and 30 additional markers in all families. Three regions (10q26.13, 11q12.3, 20p12.1) retained suggestive evidence (peak LOD scores 1.31, 1.85, 1.78; P=0.007, 0.0018, 0.0021) for linkage to tuberculosis, 3 regions (6p21.32, 17q22, 20p13) to leprosy (HLA-DQA, 3.23, P=5.8 × 10−5; D17S1868, 2.38, P=0.0005; D20S889, 1.51, P=0.004). The peak at D20S889 for leprosy is 3.5 Mb distal to that reported at D20S115 for leprosy in India. (151 words).

SLC11A1 (formerly NRAMP1) and susceptibility to visceral leishmaniasis in The Sudan

Genetic susceptibility to visceral leishmaniasis (VL) is indicated by differences in incidence and clinical phenotypes between ethnic groups in Sudan. In mice, innate susceptibility to Leishmania donovani, the etiological agent of VL, is controlled by Slc11a1 (formerly Nramp1). We therefore examined polymorphisms at SLC11A1 in 59 multicase families of VL from the high-incidence Masalit tribe in Sudan. Multipoint nonparametric analysis in ALLEGRO shows a significant linkage across SLC11A1 (Zlr scores 2.38–2.55; 0.008⩽P⩽0.012; information content 0.88). The extended transmission disequilibrium test shows biased transmission of alleles at 5′ polymorphisms in the promoter (P=0.0145), exon 3 (P=0.0037) and intron 4 (P=0.0049), and haplotypes formed by them (P=0.0089), but not for 3′ polymorphisms at exon 15 or the 3′UTR. Stepwise logistic regression analysis using a case/pseudo-control data set derived from the 59 families was consistent with main effects contributed by the intron 4 469+14G/C polymorphism. Although the two alleles for 469+14G/C lie on haplotypes carrying different alleles for the functional promoter GTn polymorphism, the latter did not itself contribute separate main effects. Sequence analysis of 36 individuals failed to identify new putative functional polymorphisms in the coding region, intron 1, intron/exon boundaries, intron 4/exon 4a, or in the 3′UTR. One novel promoter polymorphism (–86G/A) was located within a putative nuclear factor kappa B binding site that could be functional. Further work will determine whether additional polymorphisms occur upstream in the promoter, which could be in linkage disequilibrium with the intron 4 polymorphism. These studies contribute to knowledge of the role of SLC11A1 in infectious disease.

Association and linkage of leprosy phenotypes with HLA class II and tumour necrosis factor genes.

Previous analyses indicate major gene control of susceptibility to leprosy per se and the HLA class II region has been implicated in determining susceptibility and control of clinical phenotype. Segregation analysis using data from 76 Brazilian leprosy multi-case pedigrees (1166 individuals) supported a two locus model as the best fit: a recessive major gene and a recessive modifier gene(s) (single locus vs two locus model, P = 0.0007). Combined segregation and linkage analysis to the major locus, showed strong linkage to HLA class II (HLA-DQB1 P = 0.000002, HLA-DQA1 P = 0.000002, HLA-DRB1 P = 0.0000003) and tumour necrosis factor genes (TNF P = 0.00002, LTA P = 0.003). Extended transmission disequilibrium testing, using multiple affected family members, demonstrated that the common allele TNF*1 of the −308 promoter region polymorphism showed linkage and/or association with disease per se, at a high level of significance (P < 0.0001). Two locus transmission disequilibrium testing suggested susceptibility (TNF*1/LTA*2) and protective (TNF*2/LTA*2) haplotypes in the class iii region. Taken together the segregation and HLA analyses suggest the possibility of more than one susceptibility locus in the MHC.

Genetics and visceral leishmaniasis: of mice and man.

Ninety per cent of the 500 000 annual new cases of visceral leishmaniasis (VL) occur in India/Bangladesh/Nepal, Sudan and Brazil. Importantly, 80–90% of human infections are sub‐clinical or asymptomatic, usually associated with strong cell‐mediated immunity. Understanding the environmental and genetic risk factors that determine why two people with the same exposure to infection differ in susceptibility could provide important leads for improved therapies. Recent research using candidate gene association analysis and genome‐wide linkage studies (GWLS) in collections of families from Sudan, Brazil and India have identified a number of genes/regions related both to environmental risk factors (e.g. iron), as well as genes that determine type 1 vs. type 2 cellular immune responses. However, until now all of the allelic association studies carried out have been underpowered to find genes of small effect sizes (odds ratios or OR < 2), and GWLS using multicase pedigrees have only been powered to find single major genes, or at best oligogenic control. The accumulation of large DNA banks from India and Brazil now makes it possible to undertake genome‐wide association studies (GWAS), which are ongoing as part of phase 2 of the Wellcome Trust Case Control Consortium. Data from this analysis should seed research into novel genes and mechanisms that influence susceptibility to VL.