Dados do Trabalho

Título

NGS to identify genes associated with VL severity in response to IL-6 overexpression

Introdução

Visceral leishmaniasis (VL), also called kala-azar, is caused by the protozoa Leishmania infantum and L. donovani. The pro-inflammatory cytokine, interleukin-6 (IL-6), is very high in patients with VL and its plasma concentration correlates with the disease severity. Genome wide association studies (GWAS) present a powerful tool for connecting a phenotype to its underlying genetics. This method offers a hypothesis-free approach that systematically tests thousands of variants in the genome without prior knowledge about causal variants location, searching for single nucleotide polymorphisms (SNPs) that occur differentially in organisms with a particular tract versus organisms not associated with the tract.

Objetivo(s)

To verify if a host inflammatory response mark (IL-6) can be used as a trait to identify genes associated with pro-inflammatory virulence factors.

Material e Métodos

Sixty six VL patients was used for cytokine IL-6 quantification by flow cytometry. The DNA isolated from the parasites was sequenced through Illumina® Next Generation Sequencing (NGS) platform. Case-control and logistic regression analyzes were performed using PLINK software, a suite of GWAS tools. For the association analysis of SNPs with the IL-6 concentration, the strategy considered account the concentration of IL-6 in a categorical way (dichotomous). An IL-6 concentration of 300 pg/mL was estimated as the cut-off point, where the cut-off definition came from an analysis of prediction of IL-6 for death through the ROC curve (AUC = 67.1) and p-values corrected by the Bonferroni method.

Resultados e Conclusão

In the filtering referring to the adjusted p-value (p = 0.000006), only one was filtered, the SNP8239. In the logistic regression analysis, considering the multiple-test correction test, it was not possible to identify any SNP associated with IL-6. SNP8239 (p = 0.000001353) represents the exchange of a guanine for a thymine at position 1965817 on chromosome 35 in the LINF_350058100 gene, annotated as alpha-1,2-mannosyltransferase. It was present in four isolates from patients who had IL-6 greater than 300pg/ml and totally absent in patients with IL-6 less than 300 pg/ml. Leishmania spp. glycoconjugates are associated with cell signaling and can act as messengers in response to cytokines and other stimuli. Thus, it is suggested that the alpha-1,2-mannosyltransferase gene determines a pro-inflammatory virulence factor that can result in severe VL.

Palavras-chave

Leishmania infantum, cytokine, interleukin-6, GWAS, virulence factor