Discovering genes and functional pathways associated with American cutaneous leishmaniasis (ACL) caused by Leishmania brasiliensis.

Leishmaniasis is caused by protozoan parasites. There are 12 million cases world-wide, with an annual incidence is 0.9 to 1.6 million cases and a disease burden calculated by WHO as 1.98 million disability adjusted life years. American cutaneous leishmaniasis (ACL) caused by Leishmania braziliensis is among the worst forms of cuteneous leishmaniasis (CL) disease, and is associated with a clinical spectrum that ranges from one or more ulcers with raised borders to debilitating diffuse or mucosal forms of disease. Frontline drugs are those used half a century ago which are toxic and prone to development of drug resistance. There are no vaccines against leishmaniasis, making novel approaches to developing and understanding the molecular basis of how vaccines work a crucial area for continuing research.

Familial clustering of disease, racial differences in asymptomatic:disease ratios, high relative-risk ratios for siblings, and studies in mice all point to a genetic component for disease susceptibility. Previous model-based segregation analysis that allowed joint estimation of genetic and environmental effects and took gene x covariate interactions into account supports a major single gene controlling the onset of the primary cutaneous lesion caused by L. braziliensis. Many candidate gene studies have been reported to try to identify the genetic risk factors for ACL, but all have been underpowered. The only definitive way to identify genetic risk factors is to undertake well-powered genome-wide studies using large sample sizes.

Using such an approach for the visceral form of leishmaniasis we provided clear and unambiguous evidence that polymorphisms in the Class II region of the Human Leukocyte Antigen (HLA) complex are the single major genetic determinant of VL (Pcombined=2.76E-17; odds ratio=1.41; 95% confidence interval=1.30-1.52 over three cohorts from India and Brazil). This robust demonstration that the most important genetic risk factor for VL lies at the heart of eliciting host CD4+ T-cell mediated immunity against Leishmania parasites has major implications for vaccine design.

Our aim here is to emulate this major advance in understanding the visceral form of disease by undertaking a costeffective, large-scale GWAS of ACL disease. In particular, to determine whether there is the same clear cut evidence for a major role of the HLA Class II region in determining ACL disease outcome, and/or whether other important genetic risk factors play a role. To achieve this, we will (i) undertake a stage 1 discovery GWAS by genotyping 547,644 single nucleotide polymorphisms (SNPs) in 1123 ACL cases and 1122 healthy controls using the Illumina HumanCoreExome beadchip, imputing against the 1000 genomes reference panel, and using mixed models to take account of relatedness and population structure in undertaking an association analysis; (ii) undertake a stage 2 discovery GWAS by genotyping 547,644 SNPs on a second set of DNAs for 1000 ACL cases and 1000 healthy controls using the Illumina HumanCoreExome beadchip, imputing against the 1000G reference panel, and using mixed models to take account of relatedness and population structure in an association analysis; (iii) carry out detailed immunological profiling and sample banking during prospective collection of a subset of stage 2 samples; (iv) combine data for the two cohorts in a metaanalysis to provide formal evidence for replication; (v) carry out pathway analyses and functional validation of genotype against phenotype.

Overall, this proposal provides the first adequately powered study of genetic risk factors for American cutaneous leishmaniasis. Genes and pathways identified as risk factors for ACL may provide novel insights in pathogenesis of disease, seeding further research into the identification of novel therapeutic and with the potential to inform development of vaccines against this debilitating parasitic infection.

The study is designed to discover genetic risk factors for American cutaneous leishmaniasis (ACL) caused by Leishmania braziliensis in Brazil. A genome wide association study (GWAS) is proposed as follows:
1. A stage 1 discovery GWAS will be performed by genotyping 1123 ACL cases and 1122 unaffected controls for 547,644 single nucleotide polymorphisms (SNPs) using the Illumina HumanCoreExome beadchip, enhancing SNP information by imputation against a 1000 genomes reference panel, and carrying out association analysis using mixed models to control for relatedness and population substructure.
2. A stage 2 discovery GWAS will be performed by genotyping a second set of 1000 ACL cases and 1000 controls for 547,644 SNPs using the Illumina HumanCoreExome beadchip, enhancing SNP information by imputation against a 1000 genomes reference panel, and carrying out association analysis using mixed models to control for relatedness and population substructure.
3. Detailed immunological profiling and sample banking during prospective collection of a subset of stage 2 samples.
4. A meta-analysis of the data from the two discovery cohorts will be undertaken to formalise evidence for replication and enhance ability to detect weaker and/or rarer effects.
5. Second wave analyses of the GWAS output to look for evidence of pathways of genes acting as risk factors for disease will be validated functionally using data and samples acquired during phase 2 sample collection, including transcriptional profiling of lesion biopsy samples from individuals with risk genotypes.
Overall, this proposal provides the first adequately powered study of genetic risk factors for American cutaneous leishmaniasis. Genes and pathways identified as risk factors for ACL may provide novel insights in pathogenesis of disease, seeding further research into the identification of novel therapeutic targets and with the potential to inform development of vaccines against this debilitating parasitic disease.

As outlined in our data management plan, the data obtained in the course of this research project are likely to have impact that is both specific to the disease of leishmaniasis, but may also provide novel findings that impact related immunological diseases including other infectious disease and autoimmune diseases. They will also provide genotype data that can be used as a resource for other disease association studies in Brazil. To facilitate the latter, we have outlined a plan for our genotype and phenotype data to become available to the wider research community by depositing it in the European
Genome-phenome Archive (EGA) and establishing a Data Access Committee and Data Access Agreement forms as per EGA guidelines.

Our research outcomes may also directly benefit patients with disease. For example, a gene is identified as a risk factor for American cutaneous leishmaniasis might provide an immediate novel therapeutic target for which suitable drugs are currently in use for other conditions. In this case, beneficiaries may be found outside the broader academic community through partnership with industry, policy-makers, and in the primary healthcare system in Brazil. It is important that our research outcomes have the potential for major impacts on the endemic populations that are most affected by the disease, but they may also impact on related infectious and immune-related diseases, including broadening our knowledge of wound healing processes.

To maximise the potential for immediate impact, the University of Cambridge Copyright Officer at the Legal Services Office (copyright@admin.cam.ac.uk) and Cambridge Enterprise (enquiries@enterprise.cam.ac.uk) will liaise early in the project with officers of the partner institutions to lay down principles for IPR sharing, and will also facilitate negotiations that may be required with pharmaceutical partners to translate new therapeutic targets into clinical practice.

In the longer term, results of this study may impact disease control through further functional evaluation of the role of genes identified in disease pathogenesis, with the potential for novel therapies to be developed. The results may also provide important information that may impact on vaccine development, especially if the HLA region is implicated as a genetic risk factor for ACL as it was for VL.