Understanding the Mechanism of Diabetes Mellitus in LRBA-Deficient Patient
Creators: name: Hawari, Iman
Publisher: name: Manara - Qatar Research Repository
Year: 2024
Subjects: classificationCode: 42
subject: Health sciences
subject: FOS: Health sciences

description: BackgroundLRBA deficiency is associated with common variable immune dysregulation which manifests as hypogammaglobulinemia, autoimmunity, lymphoproliferation and a high susceptibility to inflammatory bowel disease in early childhood. Diabetes mellitus, growth retardation and short stature have also been reported in some patients with LRBA deficiency. LRBA deficiency induces autoimmunity through downregulation of CTLA-4, an immune checkpoint, that will result in autoreactive lymphocytes targeting autologous cells.Hypothesis1) LRBA deficiency in the patient leads to decreased surface CTLA-4 expression level and therefore immune mediated type 1 diabetes mellitus. 2) LRBA protein may be involved in the insulin biosynthesis and secretion in β-cells.AimsOur main objective is to study the molecular mechanism of diabetes mellitus in LRBA-deficient patient and to investigate if LRBA has any role in insulin biosynthesis and secretion in β pancreatic cells.MethodIn this study, the proband with infancy-onset diabetes mellitus was recruited with her family. Glutamic acid decarboxylase, insulin, protein tyrosine phosphatase and zinc transporter autoantibodies were measured. Whole genome and RNA sequencing for the proband was undertaken to identify the causative gene, and candidate mutations were confirmed using Sanger sequencing. A schematic diagram of LRBA protein with predicted domains and reported mutations of LRBA in patients diagnosed with diabetes mellitus was used to investigate clinical phenotype relation to genotype in patients with LRBA mutations. The proband’s lymphocytes were tested for their ability to be activated and express CTLA-4. The pro-inflammatory immune response of the proband’s PBMC were tested against reported immunogenic β-related peptides. LRBA was knocked down using siRNA in MIN6 cell line to investigate the role of LRBA in β-cells in more details. ResultsA novel homozygous mutation in LRBA (W1330*, c.3999 G&gt;A) was found in a child diagnosed with immunodeficiency and infancy-onset diabetes mellitus at the age of 7 months. The proband’s total lymphocytes show insignificant reduction of CTLA-4 protein level in normal condition and in response to stimulation. Looking specifically at CTLA-4 protein expression on CD8+ and CD4+ T cells, an imbalance was observed, leading to suppression of cytotoxic immune mechanism of T cells in the proband. Additionally, the proband’s lymphocytes did not show any IFN-Ɣ immunogenic response against β related peptides. Consistent with our results, blood transcriptomic analysis revealed that abundance of aggregates related to T cells or B cells (A1 &amp; A2) or inflammation (A33 &amp; A35) did not differ compared to healthy controls. However, aggregates related to CD71+ erythroid cells, which have immunosuppressive characteristic, were highly upregulated in the LRBA-deficient patients.Experimental analysis of MIN6 cell in culture revealed that in siRNA-mediated-knockdown of LRBA revealed a significant decreased intracellular proinsulin and mature insulin inside β-cells in comparison so the wild type cells. Subsequently secreted insulin levels were also lower than normal. The insulin secretion defect was confirmed after glucose stimulation insulin secretion, which lead to accumulation in the intracellular insulin levels in β cells compared to the wild type. Interestingly, insulin II mRNA abundance were significantly low in LRBA-knockdown cells, before and after glucose stimulation compared to the wild type. Knocking down LRBA did not induce immediate cell death, but lead to a reduced glucose metabolism in β cell.ConclusionTo report a novel nonsense mutation in <i>LRBA</i>. The mutation was found in a child diagnosed with diabetes mellitus in early infancy. To our knowledge, all reported cases of diabetic patients with LRBA mutation were compiled in this research. The position and protein effect of each mutation was investigated here to see if there is any genotype-phenotype relation takes place in these patients. LRBA protein deficiency was confirmed in the diabetic proband using isolated PBMCs. Our study shows overall CD3+ CTLA-4 expression was non-significantly different compared to healthy subject after stimulation with ionomycin. LRBA deficiency caused imbalanced differential CTLA-4 expression on CD4+ and CD8+ T cells leading to suppression of cytotoxic immune mechanism of lymphocytes in the patient. The low number of pro-inflammatory IFN-Ɣ cytokines against β-related proteins lead us to conclude that the beta insulin secreting cells in the LRBA-deficient proband were not targeted by the autoreactive lymphocytes of the patient. We found that LRBA is highly expressed in β-cells in mice and it modulates the biosynthesis and secretion of insulin.