The Effect Of A Novel Homozygous Variant In RasGRP1 On Disease In A Patient With An Inborn Error Of Immunity

Abstract

Background: Primary immunodeficiency diseases (PIDs) are a group of congenitally inherited heterogeneous disorders that result in the depletion or deficiency of one or more components of the immune system. RASGRP1 is a guanidine exchange factor involved in the development, differentiation, and function of lymphocytes. It regulates the activation and inactivation of RAS by adjusting the balance between GTP/GDP-bound forms. It is expressed in T cells, B cells, and innate cells, including natural killer (NK) cells. Recently, 11 patients have been reported to have inborn defects in RASGRP1 which caused severe illnesses. They presented with common clinical and laboratory features of Epstein–Barr virus (EBV) infection, EBV-related complications such as lymphoproliferative disease, lung infection, autoimmunity, decreased number of CD4+ T lymphocytes, impaired ERK/MAPK activation, and decreased T-cell proliferation in response to mitogens and antigens.

Case presentation: The patient under investigation is a 17-year-old male born to consanguineous parents with a history of Evans Syndrome. His earlier clinical history is indicative of autoimmune hemolytic anemia and decreased T-cell counts associated with recurrent infections. The early onset and severity of the clinical manifestations pointed towards an immunodeficiency.

Methods: Genomic DNA (gDNA) was purified and sequenced by Next Generation Sequencing and Sanger sequencing. Peripheral blood mononuclear cells (PBMCs) were isolated on a Ficoll-paque gradient by differential centrifugation. Immunophenotyping of PBMCs, Regulatory T cell staining, B and T cell proliferation, determination of intracellular cytokines, T-cell degranulation assay, and F-actin polymerization assay, were determined by flow cytometry. Expression of RASGRP1 and ERK phosphorylation were done by immunoblotting. Cytokine secretion was measured by Enzyme-Linked Immunosorbent Assay (ELISA). T cell function was determined using an EBV transformation assay.

Results: Immunophenotyping of PBMCs isolated from the patient revealed a very low T cell count, absent Recent Thymic Immigrants (RTEs), and normal to elevated numbers of B and NK cells. Next Generation Sequencing analysis identified a homozygous c.1396-1397insA in RASGRP1 coding sequence that resulted in the conversion of threonine to asparagine at position 466 on the RASGRP1 protein leading to a frameshift mutation (p.T466N fs*3). The c.1396-1397insA mutation abolished the expression of RASGRP1 and ERK phosphorylation downstream of TCR signaling, resulting in absent T cell activation in the form of upregulation of activation markers, proliferation and secretion of cytokines, F-actin polymerization, and inhibition of B cell transformation with EBV. B cell function was maintained.

Conclusion: We used genetic and functional approaches to identify a novel deleterious homozygous mutation in RASGRP1 and studied the effect of the mutation on protein expression and T cell development and function. Our work expands the spectrum of mutations in RASGRP1 in immunodeficiency and dysregulation.