Project

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From genome-derived knowledge to diagnostics for leprosy

English title From genome-derived knowledge to diagnostics for leprosy
Applicant Cole Stewart
Number 164174
Funding scheme Brazil
Research institution Global Health Institute EPFL SV-DO
Institution of higher education EPF Lausanne - EPFL
Main discipline Molecular Biology
Start/End 01.01.2016 - 31.12.2018
Approved amount 249'118.00
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Keywords (4)

Diagnostics; Immunogenetics; Leprosy; Pathogenesis

Lay Summary (French)

Lead
La lèpre est une maladie infectieuse chronique de l’homme résultant d’une infection par Mycobacterium leprae, un agent pathogène qui ne peut être cultivé en laboratoire. Au Brésil la lèpre (qui est difficile à diagnostiquer) représente un important problème de santé publique avec plus de 30'000 nouveaux cas détectés chaque année. Ceci en fait le pays le plus touché au monde après l’Inde. Pour ce projet, une équipe de scientifiques suisses et brésiliens vont utiliser la génétique et la génomique pour étudier la relation hôte-pathogène chez les patients atteints de la lèpre, leurs contacts familiaux et des sujets témoins.
Lay summary

Ce projet a trois objectifs principaux:

 

  1. Comparer les profils d’expression génique des personnes atteintes de la lèpre avec ceux des groupes de contrôle par séquençage de l’ARN (ARNseq). Cette approche va permettre d’identifier les gènes humains dont l’expression a été modifiée par la maladie. Les protéines codées par ces gènes pourraient servir de « biomarqueurs » qui sous-tendront le développement de nouveaux tests de diagnostic.

     

  2. Identifier les mutations génétiques qui confèrent susceptibilité ou résistance à la lèpre au moyen de comparaisons génomiques. Selon nos connaissances actuelles, de telles mutations sont susceptibles de se produire au niveau des gènes qui interviennent dans l’immunité innée, cette première ligne de défense contre l’infection. La contribution de ces changements génétiques à la lutte contre le bacille de la lèpre sera testée après leur introduction dans des lignées cellulaires par ingénierie génomique.

     

  3. Séquencer le génome de chaque souche de Mycobacterium leprae directement à partir de biopsies de peau et mettre en corrélation la séquence avec l’état pathologique et immunogénétique du patient. Ceci nous permettra d’établir l’arbre généalogique du bacille de la lèpre et de prédire si les souches sont résistantes aux médicaments, point capital car seule la polychimiothérapie permet de lutter contre la maladie.

 

Les produits attendus de ce projet de recherche intégré sont une compréhension accrue de la pathogenèse de la lèpre ainsi que le transfert bilatéral de technologie.

En outre les résultats de cette recherche vont catalyser le développement d’une nouvelle génération d’outils de diagnostic nécessaires pour aider à éliminer cette terrible maladie.

 

Direct link to Lay Summary Last update: 14.12.2015

Lay Summary (English)

Lead
Leprosy is a chronic infectious disease of humans and results from infection with Mycobacterium leprae, a pathogen that cannot be cultured in the laboratory. In Brazil leprosy, which is difficult to diagnose, remains an important public health problem since over 30,000 new cases are recorded every year; the country has the second highest burden in the world after India. In this project a team of Swiss and Brazilian scientists will use genetics and genomics to investigate the host-pathogen relationship in leprosy patients, their household contacts and control subjects.
Lay summary

The project has three main goals:

 

1. To compare the gene expression profiles of persons affected by leprosy with those of control groups by means of RNA sequencing (RNA-Seq).  This approach will identify human genes whose expression is altered by the disease.  The proteins encoded by these genes could serve as "biomarkers" that will underpin the development of new diagnostic tests.

 

2. To pinpoint genetic mutations that confer susceptibility or resistance to leprosy by means of genome comparisons.  Based on current knowledge, such mutations are likely to occur in genes that mediate innate immunity, the first line of defense against infection.  The contribution of these genetic changes to fighting the leprosy bacillus will be tested after their introduction into cell lines by genome engineering.

 

3. To sequence the genomes of each strain of Mycobacterium leprae directly from skin biopsies and to correlate the sequence with the disease state and immunogenetics of the patient.  This will allow us to establish the family tree of leprosy bacilli and also to predict whether the strains are drug-resistant, an important point as multidrug therapy is the only means of controlling the disease.

 

The expected products of this integrated research project are deeper understanding of the pathogenesis of leprosy and bilateral transfer of technology.  Furthermore, the results of this investigation will catalyze the development of a new generation of much needed diagnostic tools to help eliminate this awful disease.

Direct link to Lay Summary Last update: 14.12.2015

Responsible applicant and co-applicants

Employees

Publications

Publication
British Red Squirrels Remain the Only Known Wild Rodent Host for Leprosy Bacilli
Schilling Anna-Katarina, Avanzi Charlotte, Ulrich Rainer G., Busso Philippe, Pisanu Benoit, Ferrari Nicola, Romeo Claudia, Mazzamuto Maria Vittoria, McLuckie Joyce, Shuttleworth Craig M., Del-Pozo Jorge, Lurz Peter W. W., Escalante-Fuentes Wendy G., Ocampo-Candiani Jorge, Vera-Cabrera Lucio, Stevenson Karen, Chapuis Jean-Louis, Meredith Anna L., Cole Stewart T. (2019), British Red Squirrels Remain the Only Known Wild Rodent Host for Leprosy Bacilli, in Frontiers in Veterinary Science, 6, 1-6.
Insights from the Genome Sequence of Mycobacterium lepraemurium: Massive Gene Decay and Reductive Evolution.
Benjak Andrej, Honap Tanvi P, Avanzi Charlotte, Becerril-Villanueva Enrique, Estrada-García Iris, Rojas-Espinosa Oscar, Stone Anne C, Cole Stewart T (2017), Insights from the Genome Sequence of Mycobacterium lepraemurium: Massive Gene Decay and Reductive Evolution., in mBio, 8(5), 1-6.
Whole genome sequencing distinguishes between relapse and reinfection in recurrent leprosy cases.
Stefani Mariane M A, Avanzi Charlotte, Bührer-Sékula Samira, Benjak Andrej, Loiseau Chloé, Singh Pushpendra, Pontes Maria A A, Gonçalves Heitor S, Hungria Emerith M, Busso Philippe, Piton Jérémie, Silveira Maria I S, Cruz Rossilene, Schetinni Antônio, Costa Maurício B, Virmond Marcos C L, Diorio Suzana M, Dias-Baptista Ida M F, Rosa Patricia S, Matsuoka Masanori, Penna Maria L F, Cole Stewart T, Penna Gerson O (2017), Whole genome sequencing distinguishes between relapse and reinfection in recurrent leprosy cases., in PLoS neglected tropical diseases, 11(6), 0005598-0005598.
Red squirrels in the British Isles are infected with leprosy bacilli
Avanzi Charlotte, del-Pozo Jorge, Benjak Andrej, Stevenson Karen, Simpson Victor R., Busso Philippe, McLuckie Joyce, Loiseau Chloé, Lawton Colin, Schoening Janne, Shaw Darren J., Piton Jérémie, Vera-Cabrera Lucio, Velarde-Felix Jesùs S., McDermott Fergal, Gordon Stephen V., Cole Stewart T., Meredith Anna L. (2016), Red squirrels in the British Isles are infected with leprosy bacilli, in Science, 354(6313), 744-747.

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Conference RICAI 2017 Talk given at a conference NA 18.12.2017 Paris, France Avanzi Charlotte;
Annual Congress Fondation Raoul Follereau Talk given at a conference NA 22.09.2017 Paray le Monial, France Avanzi Charlotte; Cole Stewart;
Conference-ASM microbe 2017 Talk given at a conference NA 30.05.2017 New Orleans, United States of America Avanzi Charlotte;
Visit to FIOCRUZ Talk given at a conference NA 28.05.2017 Rio de Janeiro, Brazil Moraes Milton; Cole Stewart;
ILC 2016 – Beijing Talk given at a conference Whole genome sequencing of leprosy bacilli: from efficient DNA extraction methods to analysis of 100+ genomes 18.09.2016 Beijing, China Avanzi Charlotte;
Microbiology Society Annual Conference 2016 Talk given at a conference Leprosy – zoonosis and anthroponosis 22.03.2016 Liverpool, Great Britain and Northern Ireland Cole Stewart;
IMMEM XI Talk given at a conference Whole genome sequencing of the leprosy agent Mycobacterium lepromatosis in infected red squirrels from Scotland 09.03.2016 Estoril, Portugal Avanzi Charlotte;


Associated projects

Number Title Start Funding scheme
133797 Biology Needs Ultra High Throughput DNA Sequencing 01.06.2011 R'EQUIP

Abstract

This proposal builds on the results of a collaborative pilot project between Professor Cole's group at the Global Health Institute, EPFL, and Professor Moraes' group, the Laboratório de Hanseníase, at FIOCRUZ, that was pursued in the framework of the Brazilian Swiss Joint Research Project (grant number #0112-01). The previous investigation, entitled “Comparative Transcriptomics of Leprosy”, generated the preliminary data and main hypotheses that we now wish to test in a more ambitious, integrated manner. Leprosy is an infectious granulomatous disease caused by Mycobacterium leprae, which can result in severe disabilities in humans if diagnosed too late, this neglected tropical disease remains a global health problem and is of special significance in Brazil. Diagnosis is confounded by the many forms in which the disease can manifest and by the lack of universally effective, evidence-based diagnostic tools. At over 31,000 new cases, the incidence of leprosy in Brazil is the second highest worldwide, after India, and the number of relapse cases after multidrug therapy (MDT) is the highest in the world. In addition, the World Health Organization is concerned about multidrug-resistant leprosy in Brazil as MDT is the sole means of controlling the disease.In the present project, we will use hypothesis-driven research to underpin new interventions in support of public health measures to combat leprosy in Brazil and elsewhere. Our proposal, "From genome-derived knowledge to diagnostics for leprosy" is built around three specific aims that are interlinked and explore the same cohorts of patients and controls. In specific aim 1, for which substantial progress has already been made, we will compare the host transcriptome in whole blood using RNA sequencing (RNA-Seq) between leprosy patients and healthy controls in order to identify genes that are differentially expressed; the products of such genes could later serve as specific biomarkers for active and latent leprosy. We hypothesize that patients with active disease will display a different gene expression signature to that of healthy subjects as has been reported for tuberculosis. The protocols have been successfully established during the pilot study and the first datasets generated. However, these were too small to allow statistical significance to be reached so we will now investigate larger groups of patients and controls. Validation of the best candidate genes will involve a two-pronged approach. First, gene expression will be monitored by RNA-Seq before, during and after treatment in a small group of patients. Second, candidate gene expression will be quantified in different groups of patients and healthy individuals using RTqPCR. Expression levels of the newly validated candidate genes will also be measured in a population of household contacts and the robustness of the approach challenged by measuring expression of the candidate genes in a blind test. The expected outcome is knowledge about genes/proteins that differentiate between disease, exposure and cure. This knowledge could later be translated into a diagnostic application such as an ELISA test that measures the levels of a specific protein in human blood or a point-of-care diagnostic test.Specific aim 2 is linked to specific aim 1 and involves genotyping the patients and their contacts by means of genome-wide association studies (GWAS) in order to identify single nucleotide polymorphisms (SNP) in the host genome or signatures in expression of genes and pathways. SNPs will also be surveyed in two household contact populations, one who developed the disease and one who did not. From the literature and our own results, we expect to uncover SNPs in genes for innate immunity such as PARK2, encoding the ubiquitin ligase Parkin, or the NOD2 pathway (NOD2, RIPK2, TNFSF15, and LRRK2). In parallel, based on the results of the integrative approach, we will analyze the function of these genes using an ex vivo system. Genome editing will be undertaken using the CRISPR/Cas9 system to introduce these mutations into human THP-1 cells. The THP-1 parent and the resultant mutant cell lines will then be infected with live M. leprae and the host response compared either by RNA-Seq or by using highly sensitive ELISA to measure the levels of cytokines/chemokines associated with innate immunity, such as the type I interferons. In this manner we will confirm causality between the SNPs discovered and susceptibility/resistance to leprosy. Furthermore, this approach will allow us to rank the candidate genes in terms of their power and functional importance. Once again, this information could underpin a diagnostic test for leprosy or exposure to M. leprae.Finally, to complete this investigation in Specific aim 3, we will attempt to correlate the genome sequence of the M. leprae strain from each patient with his/her disease state, haplotype and immunogenetics. We have recently optimized a DNA extraction method, which includes a step for host DNA depletion, that allows us to obtain near complete genome sequences from human skin biopsies in a cost-effective manner. From the genome sequences, we will then deduce the strain genotype, establish phylogenies and predict the drug susceptibility or resistance profiles. All the genome-derived information will be centralized in a web-based relational database that also contains details of the patient's case history and demographics.Based on the successful outcome of our earlier collaboration, we are fully confident that we can complete all three specific aims in the allotted timeframe as the foundations for the investigation have been laid and the cohorts of patients and controls established or identified. We anticipate that our integrated approach will lead to deeper understanding of the pathogenesis of leprosy and to bilateral transfer of technology. Furthermore, the subsequent translation of the results of our investigation into diagnostic tools can be envisioned and this may provide a response to an unmet medical need both in Brazil and in other countries where leprosy remains endemic.
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