Forward genetics of human immunity: the molecular basis of a novel form of combined immunodeficiency

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Keywords: Genetics, immunology, immunodeficiencyAbstract: Inborn errors of the immune system can lead to serious infection, autoimmunity and cancer in young children. These primary immunodeficiencies represent important models through which to understand the function of individual genes and pathways in human immunity. Next generation sequencing techniques have powerful potential to reveal disease-causing variants in novel genes but mechanistic studies are required to maximise the benefits for both scientific understanding and clinical care. The aim of this studentship will be to determine the underlying molecular basis for a newly recognised form of combined immunodeficiency. The student will examine how selected candidate variants segregate with disease in the affected families, their effect on expression and function of the related protein and the mechanism whereby such changes cause disease. Bioinformatic analysis of whole exome sequencing data from affected children will be achieved through a combination of in house, proprietary and publicly available software tools that are already in established use within the host laboratory. Having filtered this down to rare, predicted deleterious variants with the expected pattern of inheritance, the student will assess the goodness of fit between gene and phenotype. For example, they will interrogate published data on gene expression and function, examine the degree of evolutionary conservation of any mutated residue, map variants to protein structure where known and compare with known variants eg in murine homologues or somatic mutations in cancer. Selected variants will be taken forward for genetic and functional validation. To confirm and filter candidate variants further, segregation will be assessed by polymerase chain reaction (PCR) amplification across each variant, using genomic DNA from unaffected and affected family members as template, followed by Sanger sequencing of PCR amplicons (performed commercially). The effects of the variant on mRNA and protein abundance will be assessed in patient cells where possible (by quantitative RT-PCR and immunoblotting +/- immunofluorescence, respectively). Generally we will have access to patient fibroblasts and peripheral blood mononuclear cells, and often B lymphoblastoid cell lines. Alternatively the mutation will be modelled by site-directed mutagenesis of a mammalian expression construct and studied in a recombinant system. For missense mutations with residual protein expression, the structural effect of amino acid substitutions will be modelled into known molecular structures - this may give insight into their effect on protein function. Further studies will be tailored to the protein of interest to delineate functional effects on the affected pathway, whether in patient-derived cells or model systems generated by gene knock down, genome editing or even small molecule inhibitors in cultured cells. Training will be provided in core techniques (tissue culture, transfection, flow cytometry, immunoblotting, imaging, ELISA, qPCR etc). We have access to state of the art facilities including bioimaging, flow cytometry, a new single cell genomics facility offering CyTOF and Illlumina NextSeq for on-site transcriptomics etc.