Streptococcus pneumoniae (pneumococcus) is a commensal organism of the human nasopharynx that causes common benign infections of the upper respiratory tract. In rare cases, it may cause invasive disease, such as septicemia and meningitis. Invasive pneumococcal disease (IPD) typically occurs in children under two years of age.
We hypothesize that invasive pneumococcal disease (IPD) in childhood is favored by unknown Mendelian disorders of the immune system, at least in some children. We intend to develop hypothesis-generating, genome-wide approaches for identifying pathways normally involved in immunity to pneumococcus but deficient in patients with IPD. We will combine genetic and genomic approaches to achieve this goal.
We aim 1) to recruit children with unexplained IPD, including children born to consanguineous and/or multiplex kindreds; 2) to identify novel primary immunodeficiencies in children with IPD, using a genetic approach (gene positional mapping); and 3) to identify immunological pathways deficient in patients with IPD, using a genomic approach (genome-wide immune reactivity profiles).
A cohort of 45 children with unexplained, “idiopathic” IPD, with no detectable immunological defect, has already been set up, forming the core study population. Over the next three years, an additional 55 children will be recruited, resulting in the enrollment of 100 children with IPD in total. The genetic approach will be based on the genotyping of polymorphic markers (SNPs) throughout the genome (genome-wide scan) in a series of unrelated kindreds, each consanguineous and containing at least one child with IPD with no detectable cellular phenotype.
The genomic approach will investigate the immune responses of patients with IPD in a blood re-stimulation assay. Blood samples, unstimulated and stimulated with killed S. pneumoniae, TNF-a, IL-1b and various Toll-like receptor agonists, will serve as templates for mRNA extraction and genome-wide expression profiling. A reference dataset will be generated, using samples from patients with known deficiencies (IRAK4 -/-; NEMO -/-; MYD88 -/-), and profiles will be generated under the same experimental conditions for blood obtained from patients with IPD carrying no known mutation. The mutations identified will be validated at the mRNA (northern blot, RT-PCR) and protein (western blot, immunoprecipitation, flow cytometry) levels, and by gene transfer experiments.