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Genetic Control of Cutaneous Leishmaniasis 1Noyes, HA: 3Arana, FE; 3Rizzo, N; 1Kemp, SJ; 2Hutchinson, IV; 2Pravica V; 1Daly, D; 3Arana, BA; 1School of Biological Sciences, University of Liverpool, L69 7ZB, UK 2School of Biological Sciences, University of Manchester, M13 9PT, UK 3Medical Entomology Research and Training Unit, University del Valle, Guatemala. Abstract Sample Collection and Processing Differences in expression of cytokines involved with the Th1 and Th2 responses are associated with single nucleotide polymorphisms (SNP) in the genes that encode them.. We have genotyped SNP in a panel of genes for cytokines that are known to be important in controlling the immune response. Samples were collected from the following groups: 1) Patients with chronic cutaneous leishmaniasis of more than four months. 2) Subjects with a positive Montenegro skin test but no history of disease. These individuals were expected to be resistant to the disease but not infection. 3) Individuals from the same endemic area with no history of disease and a negative skin test. This is expected to be a heterogeneous group who may or may not have been challenged Significant differences were found in allele frequencies in the TNF-beta and IL4 genes. Samples were collected in the Department of Peten in Guatemala. The Peten is an agricultural frontier area where forest has been cleared for subsistence agriculture over the last forty years. Cutaneous leishmaniasis (CL) due to Leishmania mexicana and L. braziliensis is endemic in this region. Samples were collected from patients at the regular clinics run by the University del Valle (Guatemala) in the town of Poptun in Peten. These clinics are announced on the local radio and provide a free service to all who attend. Lesion scrapings for parasite identification and a 10ml blood sample are obtained from all patients who have lesions consistent with cutaneous leishmaniasis. A Montenegro skin test (MST) is applied to all patients. Parasites are identified by nested PCR (Noyes et al. 1998). Patients with a clinical diagnosis of CL were treated with the WHO recommended dose of Glucantime. Samples were collected controls by establishing clinics in communities which were known to be endemic for CL. All members of the community were offered a test for anaemia and an MST skin test. 10ml blood samples were taken from all volunteers over the age of 15. Samples were also collected from any cases of CL that were discovered. DNA was prepared from blood by ammonium chloride lysis of red cells and salt precipitation of DNA. Cytokine alleles were identified in IL-6, IL-10, TNF-β, TNF-α, IFN-γ, IL4, IL15, TGF-beta by ARMS-PCR (Perrey et al 1998). Additional SNP in GranzymeB and CCL5 were genotyped by SnapShot on and ABI Prism 3100. A Chi squared test was used to identify significant differences between observed and expected allele frequencies and genotype frequencies of individual loci. SVD Clustering of arrays Symptomatic; 18 hours Asymptomatic; 18 hours Symptomatic; 24 hours Asymptomatic; 24 hours Second Principal Component Although microarrays are best known for gene expression analyses they are also a powerful resource for identifying clusters of samples and have been widely used for classifyin cancer cases. PBMC were taken from 30 MST+ve subjects with a history of disease and 30 MST+ve subjects with no history of disease. PBMC were stimulated with conA and harvested at 18 and 24 hours post stimulation. RNA was prepared from each sample and combined into pools of five RNA samples. Each pool was labelled with cy3 and cy5 dyes to provide a dye flip replicate and hybridised to oligonucleotide microarrays representing approximately 20,000 human genes (HGMP) using a common reference. The normalised data was clustered using Single Value Decomposition in the MaxD microarray data analysis package. After clustering the slides four groups could be identifying corresponding to the cells from the two groups of patients that had been stimulated for two lengths of time. The two groups of patients were separated on the first principal component and the two times were separated on the second principal component. Although the two groups of subjects overlapped a Student T-test indicated that the difference between them was highly significant (p=0.00059). The subjects had been selected at random irrespective of ethnic group, a Pearson correlation coefficient indicated that there was no correlation between ethnic group and co-0rdinates on the first principal component (r2 PCA1 = 0.027; (r2 PCA2 = 0.008). IL4 allele frequencies 1 P<0.001 0.8 Diseased 0.6 Infected 0.4 Uninfected 0.2 0 Indigenous Ladino TNF beta P<0.05 0.8 0.7 0.6 0.5 MST+ve w ith lesion 0.4 MST+ve no lesion MST-ve no lesion 0.3 0.2 0.1 Significant differences were found at the IL4-590 and TNF-b loci between MST+ve subjects with no history of disease and both subjects with a history of active disease and subjects with no history of disease. In the case of IL4 this difference was only observed in the “Ladino” population of immigrant descent, and there was no significant difference in allele frequencies among the different patient groups of Indigenous descent. In the case of TNF-beta the two ethnic groups had opposite trends. If confirmed these results would indicate that these cytokines might play a role in conversion on the skin test but have no effect on the development of disease. 0 Indigenous Ladino Discussion Genetic risk factors for susceptibility to cutaneous leishmaniasis First Principal Component References and Acknowledgements We gratefully acknowledge the participants in this study and Milo Henstermann and all the others who conducted the field work. This work was funded by the Wellcome Trust. Davies, C.R. and Gavgani, A.S.M. (1999) Age, acquired immunity and the risk of visceral leishmaniasis: a prospective study in Iran. Parasitology 119:247-257. Noyes, H.A., Reyburn, H., Bailey, J.W., and Smith, D. (1998) A nested-PCR-based schizodeme method for identifying Leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan. Journal of Clinical Microbiology 36:2877-2881. Perrey, C., Pravica, V., Sinnott, P.J., and Hutchinson, I.V. (1998) Genotyping for polymorphisms in interferongamma, interleukin-10, transforming growth factor-beta 1 and tumour necrosis factor- alpha genes: a technical report. Transplant Immunology 6:193-197 Turner, D.M., Williams, D.M., Sankaran, D., Lazarus, M., Sinnott, P.J., and Hutchinson, I.V. (1997) An investigation of polymorphism in the interleukin-10 gene promoter. European Journal of Immunogenetics 24:1-8. There is no evidence of any difference in susceptibility to leishmaniasis between ethnic groups in Guatemala, with a prevalence of history of leishmaniasis about 10% in both the indigenous and immigrant populations. However there was some evidence that the two groups might have different risk factors for conversion on the skin test. Most notably at the IL4-590 locus where there was a significant risk of conversion on the skin test associated with the C allele in the indigenous group but not the Ladino group. It is not uncommon for associations found in one ethnic group to be missing in another group. However , the sample sizes in this study were small and it will be important to determine if the results observed here are reproduced in other samples form the same population as well as in other ethnic groups to determine whether these observations were a real risk factor or whether they are artefacts of small sample sizes. Genetic basis for conversion on the skin test? Since the micoarray study was undertaken on cells stimulated with ConA rather than a Leishmania antigen the results provide evidence that there is a genetic component to the risk of conversion on the skin test. This may have important implications for the interpretation of the results from this widely used diagnostic and epidemiological test.