Clinical, Immunological and genetic study of patients with primary humoral immunodeficiency

Primary humoral immunodeficiencies are characterized by defect in antibody function, impaired antibodies production or both, ranging from Specific Polysaccharide Antibody Deficiency (SPAD) to agammaglobulinemia. In this project, we will focus on Primary Antibody Deficiencies (PAD) involving the BCR signaling pathway (especially PLCγ2/Calcineurin/NFAT and PI3K-Akt pathways). We hypothesize than PLCG2, PIK3CD and IRF2BP2 play a key role in B cell development, proliferation, differentiation and function but also in other immune cells as they have been previously associated with PAD. We also hypothesize that they play a major role in innate immunity, especially in inflammatory response. During this project, we will analyse in depth the mechanisms of these genes in vitro in immune cells. This will allow us to understand what exactly their roles are and what their impact on the immune system is. Meanwhile, we will perform whole exome sequencing in patients presenting with primary humoral deficiencies without any confirmed genetic diagnosis to uncover new mutant genes or new variants in known genes that could lead to PAD. We have already collected data and samples of 27 patients in 11 different families with PAD carrying a variant in IRF2BP2, each family carrying a different new variant. An already established collaboration with NIH (Dr Hsu and Dr Holland) allowed us to get access to more samples and more different gene variants. We are currently collecting data and samples from families with PLCG2 and PI3KCD variants. We already studied protein expression of two different variants in IRF2BP2 in patient’s cells (Peripheral Mononuclear Blood Cells – PBMCs) by western blot, showing an overexpression of the protein of interest in patients. We also already evaluated B cell activation, proliferation and differentiation in plasmablasts starting from patients B cells for three IRF2BP2 variants. We could show that patients carrying these variant have a defect in activation, proliferation and differentiation in their B cells and we could confirm the defect in antibody production. We plan to overexpress the variants of studied families and variants from database (as gNomad) in HEK293T cells and in immune cells (especially B and T cells) to evaluated protein expression. We will also evaluate gene expression by transcriptome analysis (RNASeq) and Nanostring. B cell function and development will be evaluated starting from naïve B cells. T cell activation, proliferation and differentiation will also be evaluated. To validate the impact of patient’s mutations on the immune system, transduction of studied mutations in healthy controls will be performed. We will evaluate B and T cells activation, differentiation and proliferation on transduced cells with the assays described previously. To validate variants as being pathogenic, we will transduce wild-type gene in patient’s cells and will evaluate the “rescue” impact on the assays described above by repeating the functional assays on transduced B and T cells.