The Contribution of Two Mutations in STX11 and LCP2 to the Disease in a Patient with an Inborn Error of Immunity

Abstract

Inborn Errors of Immunity (IEI) or Primary Immunodeficiencies Diseases (PIDs) comprise 406 disorders originating from 430 defected genes recorded to date. While generally monogenic, mounting evidence suggests the contribution of more than one gene-defect in the development of IEI. The genetic defects underlying the development of IEI is translated into protein loss- or gain-of-function, resulting in an impaired immune system. Severe Combined Immunodeficiency (SCID) is one of the most severe forms of IEI that affects infants at birth and is characterized by defective development and/or function of T cells (T-B+NK+ SCID), altering cell-mediated immunity. Individuals with SCID may additionally suffer from an altered humoral immunity, characterized by the lack or sparse number of B cells (T-B-NK+ SCID) and/or NK cells (T-B+NK- SCID). I am studying a patient with IEI bearing homozygous variations in both Syntaxin-11 (STX-11) and Lymphocyte Cytosolic Protein 2 (LCP2). STX-11 is implicated in the control of T/NK cell function by regulating the fusion of cytotoxic granules with the plasma membrane of cytotoxic cells, and the subsequent exocytosis of lytic granules that mediate target cell killing. On the other hand, LCP2 is an adaptor protein essential for T cell activation and TCR signal transduction. The aim of my thesis is to determine the dual contribution of the variants in both LCP2 and STX-11 to the disease of the patient. I am using a wide variety of immune techniques, including surface and intracellular flow cytometry, T and B cell proliferation using the dye-dilution method, immunoblotting, RNA/DNA extraction, PCR amplification for sequencing, and F-actin polymerization. The patient suffers from severe inflammations and infections. Using next generation sequencing, we identified a truncating mutation in STX-11 that abolished protein expression, as determined using immunoblotting. This mutation is most likely responsible for the inflammatory disease in the patient, as it was previously described in patients with Hemophagocytic Lymphohistiocytosis (HLH). A second variation was identified in LCP2; thus, we hypothesized that this variation might affect T cell signaling in the patient. The mutant LCP2 levels are decreased by 85% in the patient compared to the controls, and her T cells fail to proliferate after cross-linking the T cell receptor (TCR) suggesting that the LCP2 variant might contribute to the disease. We obtained blood from four apparently healthy siblings of the patient and performed detailed immunophenotyping and T cell functional assays that showed normal T, B, and NK cell percentages in the siblings. However, three of the siblings showed absent T cell proliferation after cross-linking their T cell receptor, suggesting defective signaling downstream of the T cell receptor. In my future work, we will sequence the DNA of the siblings to identify if any carries the LCP2 variation and determine its effect on RNA/protein expression. If one of them is homozygous for the variation in LCP2, we will repeat the T cell functional studies on them. In addition, we will also use LCP2-deficient Jurkat cells reconstituted with wild-type and mutant LCP2 to determine if it can correct their function. This study will determine the putative effect of a novel variant in LCP2 on the function of T cells and highlight the combined effect of two gene-defects on the disease in a patient with IEI.