Vici syndrome is a complex multisystem disorder characterized by congenital malformations of the brain, cataracts, skin hypopigmentation, progressive damage of cardiac function and muscle, immune deficiency. Affected children do not respond to vaccination, have low levels of antibodies in the blood and suffering from severe and frequent infections. The disease is caused by a mutation of the gene EPG5, which encodes a protein essential for the trafficking and function of intracellular vesicles.
Cells use vesicles to eliminate uwanted material and remove dead cells. The unwanted material is incorporated into a vesicle and then merged with lysosomes containing proteins that digest the contents to be eliminated (autophagy).
This system is essential already during the embryonic stage and then for life. Therefore, its failure causes a disease involving various organs and systems. Since infections are frequent in patients with Vici Syndrome, we think that EPG5 plays a role in the immune system:
In this project i propose to assess whether EPG5 is necessary for cells to capture and degrade microorganisms and foreign particles, a necessary step to start the immune reaction. To do this i use cells from patients and healthy controls with fluorescent molecules to study the formation and trafficking of vesicles and evaluate the activation of immune cells by measuring the levels of antibodies and cytokines.
Other metabolic diseases and multisystem syndromes, often also associated with immunodeficiency, are in fact caused by genes involved in trafficking, merge or release of vesicles in the cell. My study is a prototype, intended to clarify the underlying mechanisms of a defect in a specific immune complex disease. The results will be used to develop therapies appropriate to fight at least in part the symptoms and risks in patients with multisystem genetic disease.
Table of Contents
Chapter 1 – INTRODUCTION
1.1) Vici Syndrome
1.2) EPG5
1.3) Autophagy and endocytosis
1.4) Pathogenesis of Vici Syndrome
1.5) Toll-like protein receptors (TLRs)
Chapter 2 – AIM OF THE WORK
Chapter 3 – MATERIALS AND METHODS
3.1) Materials
3.1.1) Patients clinical data
3.1.2) Cell Cultures
3.2) Methods
3.2.1) Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
3.2.2) Generation of a lymphoblastoid cell lines
3.2.3) PBMCs stimulation with TLR2-9 agonists
3.2.4) Fibroblast stimulation with CpG or IL-1β and NF-kBp65 translocation
3.2.5) CpG-FITC internalization in lymphoblastoid cell lines (LCLs)
3.2.6) Immunofluorescence
3.2.7) Transient gene silencing by siRNA
3.2.8) Transient gene transfection by lipofection
3.2.9) Transient gene transfection by electroporation
3.2.10) Extraction of genomic DNA
3.2.11) Extraction and quantification of the RNA
3.2.12) Reverse transcription and amplification (RT-PCR)
3.2.13) Electrophoresis, extraction from the agarose gel and sequencing of PCR products
3.2.14) Amplification and quantification by real-time PCR
3.2.15) Protein extraction and quantification
3.2.16) Electrophoresis of proteins by SDS-polyacrylamide gel
3.2.17) Western blotting
3.2.18) Plasmid constructs and transformation of bacterial cells
3.2.19) Preparation of plasmid DNA
3.2.20) DQ-BSA internalization
Chapter 4 – RESULTS
4.1) Molecular genetic analysis
4.2) Analysis of the effects of mutations on the transcribed EPG5
4.3) Analysis of the effects of the mutations on protein EPG5
4.4) Stimulation of PBMC in vitro with TLR2-9 agonists
4.5) Detection of NF-kB p65
4.6) Analysis of intracellular transport of DQ-BSA
4.7) Analysis of intracellular transport of CpG (EAA1, LAMP1, LAMP2)
4.8) Internalization of CpG-FITC in the patient’s and control fibroblasts
4.9) Transient silencing of EPG5 and internalization of CpG-FITC
4.10) Cellular localization of EPG5 in HEK293T cell lines
Chapter 5 – DISCUSSION
Objectives and Research Themes
This thesis aims to decipher the underlying molecular mechanisms of immune dysfunction in Vici syndrome. Specifically, the research investigates how mutations in the EPG5 gene impact the autophagy and endocytic pathways, leading to the characteristic immunodeficiency observed in affected patients.
- Investigation of molecular defects caused by EPG5 mutations.
- Analysis of the endocytic pathway and late endosome-lysosome fusion.
- Evaluation of B cell response and TLR9 signaling alterations.
- Study of the impact of EPG5 gene silencing and overexpression on cellular phenotype.
Excerpt from the Book
1.1) Vici Syndrome
Vici syndrome (OMIM # 242840) is a rare genetic multisystem disorder characterized by five main clinical signs: 1) Agenesis of the corpus callosum (ACC) (total or partial lack of the truncus or “body” of the corpus callosum, connecting the right and left hemisphere of the brain); 2) Cataract (progressive clouding of the lens inside the eye which lead to a decrease in vision); 3) cardiomyopathy(cardiac changes pronounced in the left compared to the right ventricle); 4) Immunodeficiency (functional deficiency of the immune mechanisms); 5) hypopigmentation (variable from total albinism in a partial pigmentation of the retina). (Figure 1)
Vici syndrome is a multisystem disorder that is transmitted as an autosomal recessive trait. The prevalence is unknown at the time and only 20 cases have been reported worldwide (Cullup et al, 2014).
History of Vici Syndrome
In 1988, Dionici Vici et al. described for the first time the clinical case of two brothers with a syndrome of malformations with agenesis of the corpus callosum, cutaneous hypopigmentation, bilateral cataracts, cleft lip (cheiloschisis), cleft palate (palatoschisis), thymus hypoplasia and combined immunodeficiency (Dionisi Vici et al., 1988). The clinical history of the two patients was characterized by severe psychomotor retardation, epilepsy, recurrent infections of the respiratory tract, and chronic mucocutaneous candidiasis. The immunological analysis showed a severe lack of T4+ lymphocytes and IgG2 serum immunoglobulin deficiency. The two brothers died of bronchopneumonia at the age of 2 and 3 years. The clinical and histopathological features seemed to indicate a defect in the organization of the embryonic central nervous system (agenesis of the corpus callosum, hypoplasia of the cerebellar vermis), and of the immune system.
Summary of Chapters
Chapter 1 – INTRODUCTION: Provides a comprehensive overview of Vici syndrome, the EPG5 gene, and the fundamental roles of autophagy, endocytosis, and Toll-like receptors in human immunity.
Chapter 2 – AIM OF THE WORK: Outlines the study's goal to investigate how EPG5 mutations disrupt molecular pathways, specifically addressing the observed immunodeficiency through autophagy and endocytosis analysis.
Chapter 3 – MATERIALS AND METHODS: Details the experimental procedures including patient clinical data, cell culture maintenance, gene silencing, immunofluorescence, and biochemical techniques like Western blotting and RT-PCR.
Chapter 4 – RESULTS: Presents findings from genetic analysis and cellular experiments, demonstrating that EPG5 mutations lead to defective fusion of late endosomes and lysosomes, impairing TLR9 signaling.
Chapter 5 – DISCUSSION: Synthesizes the results to propose Vici syndrome as a paradigm for multi-system disease caused by defective vesicular trafficking, highlighting the importance of EPG5 in cellular homeostasis.
Keywords
Vici syndrome, EPG5, Autophagy, Endocytosis, Immunodeficiency, Toll-like receptors, TLR9, Lysosomal fusion, Genetic mutations, Molecular biology, Cell signaling, NF-kB, Congenital disorder, Vesicular trafficking, Clinical immunology
Frequently Asked Questions
What is the core focus of this research?
The work investigates the molecular mechanisms behind the immune deficiency observed in patients with Vici syndrome, specifically linking the EPG5 gene to defects in autophagy and endocytosis.
What are the primary thematic areas explored?
The study centers on Vici syndrome pathophysiology, the role of the EPG5 gene, autophagy-endocytosis pathway integration, and Toll-like receptor (TLR) signaling dysfunction.
What is the central research question?
The research asks why defects in EPG5 expression cause immunodeficiency and seeks to define the role of this gene in the immune response and intracellular transport pathways.
Which scientific methods were employed?
Methodologies included RT-PCR, quantitative real-time PCR, Western blotting, confocal immunofluorescence, flow cytometry, gene silencing (siRNA), and plasmid-based transient transfection.
What does the main body cover?
It covers clinical descriptions, materials and protocols, genetic analysis, expression studies, and detailed experimental demonstrations of altered endocytic and autophagic transport in patient cells.
What key terms characterize the study?
Vici syndrome, EPG5, autophagy, endocytosis, and immunodeficiency are the most significant descriptors of the work.
How does Vici syndrome affect B cells?
Patients exhibit a reduction in memory B cells and a failure of B cells to respond to CpG, a ligand for the TLR9 receptor, leading to reduced antibody production.
What is the functional defect regarding the EPG5 gene?
The study demonstrates that EPG5 is essential for the late steps of the endocytic pathway, specifically the fusion of late endosomes with lysosomes, which is necessary for TLR9 signaling and autophagy.
- Citar trabajo
- Evangelos Axiotis (Autor), 2015, Deciphering the Mechanism of Immune Dysfunction in Vici Syndrome, Múnich, GRIN Verlag, https://www.grin.com/document/295415
