Host and pathogen genomics of severe pediatric infections

Infectious diseases are among the leading causes of human morbidity and mortality, with the greatest burden felt in the pediatric population. For any infectious disease, only a fraction of the exposed individuals develop clinical symptoms. These inter-individual differences can be due to variation in pathogen virulence or in host susceptibility. The recent advent of high-throughput sequencing (HTS) technology has enabled studies of both human and pathogen genetic factors that have the potential to influence infectious diseases pathogenesis and alter clinical presentation. In this thesis, I present a set of genomic studies that used HTS to dissect the genetic basis of life-threatening infections with Pseudomonas aeruginosa (P. aeruginosa) and respiratory syncytial virus (RSV). This work provides conclusive evidence for the role of rare human genetic variants in susceptibility to life-threatening P. aeruginosa and RSV infections in previously healthy children. Furthermore, in an attempt to determine the role of viral genetic factors in severe presentations of RSV infection, I established a framework for exploring RSV genetic variation using HTS technology and bioinformatic analysis. Together, theses studies demonstrate that current genomic technology, bioinformatic analysis and functional follow-up have the potential to give us novel insight into the molecular basis of host-pathogen interactions and infectious disease pathogenesis.

Genomics and transcriptomics of the Mycobacterium tuberculosis complex

Swapna Uplekar

The goal of eliminating tuberculosis (TB) by 2050 depends on the development of improved TB diagnostics, drugs and vaccines. Advances in these areas require a deep understanding of the disease and its causative agent, Mycobacterium tuberculosis (M. tb). Mycobacterial species that cause TB in humans and other mammalian hosts are grouped within the M. tb complex. Development of powerful technologies such as next-generation sequencing and microarrays opened up new avenues for comparative and functional genomics of the M. tb complex. Due to the large and increasingly complex datasets generated from these technologies, the bottleneck in biological investigation has shifted from data generation to analysis. The objectives of this thesis were to establish and employ strategies for the analysis, integration, and interpretation of high-throughput sequencing and microarray datasets using a range of bioinformatics and statistical tools. In the area of comparative genomics, we assessed the genetic diversity in the M. tb complex using various methods, such as SNP (single nucleotide polymorphism) genotyping, automated Sanger sequencing and next-generation sequencing. In a study comparing the genomes of the virulent M. bovis and M. bovis BCG vaccine strains, we identified a set of SNPs that were common to all BCG strains, and could provide novel insights on the molecular basis of BCG attenuation. In another study, we surveyed the genetic variation in the highly immunodominant esx gene family among clinical isolates of M. tb and identified sequence polymorphisms in known T- cell epitopes on Esx proteins that could affect their immunogenicity. We exploited the power of next-generation sequencing to detect sequence variation among M. tb strains that could result in phenotypic differences. By comparing the genomes of drug-resistant mutants with the sensitive wild-type strain we were able to identify the target of the anti-TB drug, pyridomycin. Using a similar approach we identified a mutation that makes M. tb strains incapable of producing PDIMs (phthiocerol dimycocerosates), which are cell wall associated lipids involved in M. tb virulence. In the area of functional genomics, we mapped genome-wide binding sites for transcription factors using chromatin immunoprecipitation followed by hybridization to microarrays (ChIP-on-chip) or sequencing (ChIP-seq), and performed transcription profiling by means of high-throughput cDNA sequencing (RNA-seq). We carried out a comprehensive study to characterize the whole transcriptome of M. tb in exponential and stationary phases of growth, and understand the genome-wide dynamics of two key components of the transcription machinery, namely, RNA polymerase and NusA. By systematic integration of the ChIP-seq and RNA-seq data, we identified a set of transcription units (TU) in the M. tb genome, and mapped their putative promoters. Analysis of RNAP and NusA binding across the promoter and body of TUs and their correlation with transcription uncovered new functional aspects of the transcriptional complex in M. tb. We also exploited the ChIP-on-chip and ChIP-seq technologies to define the regulon of the M. tb sigma factor F, and gain a better understanding of the regulatory role of the nucleoid associated protein, EspR. Altogether, this thesis has improved our knowledge of the evolution, physiology and virulence of the M. tb complex. In addition, we have established next generation sequencing as a powerful tool for comparative and functional studies, with potential applications in the clinical setting.

EPFL2012

Exome Sequencing Reveals Primary Immunodeficiencies in Children with Community-Acquired Pseudomonas aeruginosa Sepsis

Samira Asgari, Istvan Bartha, Christoph Berger, Jacques Fellay, Richard Wong

One out of three pediatric sepsis deaths in high income countries occur in previously healthy children. Primary immunodeficiencies (PIDs) have been postulated to underlie fulminant sepsis, but this concept remains to be confirmed in clinical practice. Pseudomonas aeruginosa (P aeruginosa) is a common bacterium mostly associated with health care-related infections in immunocompromised individuals. However, in rare cases, it can cause sepsis in previously healthy children. We used exome sequencing and bioinformatic analysis to systematically search for genetic factors underpinning severe P. aeruginosa infection in the pediatric population. We collected blood samples from 11 previously healthy children, with no family history of immunodeficiency, who presented with severe sepsis due to community-acquired P. aeruginosa bacteremia. Genomic DNA was extracted from blood or tissue samples obtained intravitam or postmortem. We obtained high-coverage exome sequencing data and searched for rare loss-of-function variants. After rigorous filtrations, 12 potentially causal variants were identified. Two out of eight (25%) fatal cases were found to carry novel pathogenic variants in PID genes, including BTK and DNMT3B. This study demonstrates that exome sequencing allows to identify rare, deleterious human genetic variants responsible for fulminant sepsis in apparently healthy children. Diagnosing PIDs in such patients is of high relevance to survivors and affected families. We propose that unusually severe and fatal sepsis cases in previously healthy children should be considered for exome/genome sequencing to search for underlying PIDs.

Frontiers Media Sa2016

Human genomics of acute liver failure due to hepatitis B virus infection: An exome sequencing study in liver transplant recipients

Samira Asgari, Nimisha Chaturvedi, Jacques Fellay, Christian Hammer, Petar Scepanovic

Acute liver failure (ALF) or fulminant hepatitis is a rare, yet severe outcome of infection with hepatitis B virus (HBV) that carries a high mortality rate. The occurrence of a life-threatening condition upon infection with a prevalent virus in individuals without known risk factors is suggestive of pathogen-specific immune dysregulation. In the absence of established differences in HBV virulence, we hypothesized that ALF upon primary infection with HBV could be due to rare deleterious variants in the human genome. To search for such variants, we performed exome sequencing in 21 previously healthy adults who required liver transplantation upon fulminant HBV infection and 172 controls that were positive for anti-HBc and anti-HBs but had no clinical history of jaundice or liver disease. After a series of hypothesis-driven filtering steps, we searched for putatively pathogenic variants that were significantly associated with case-control status. We did not find any causal variant or gene, a result that does not support the hypothesis of a shared monogenic basis for human susceptibility to HBV-related ALF in adults. This study represents a first attempt at deciphering the human genetic contribution to the most severe clinical presentation of acute HBV infection in previously healthy individuals.

2019

Genomics and transcriptomics of the Mycobacterium tuberculosis complex

Swapna Uplekar

EPFL2012