WHOLE EXOME SEQUENCING IN VACCINE-INDUCED THROMBOTIC THROMBOCYTOPENIA

The global rollout of COVID-19 vaccines, particularly those based on adenoviral vectors (such as ChAdOx1 nCoV-19 and Ad26.COV2.S), has been associated with rare but serious adverse events. Among these, Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT) has emerged as a rare clinical syndrome resembling heparin-induced thrombocytopenia (HIT), but occurring in the absence of heparin exposure. While the pathophysiology of VITT involves antibodies against platelet factor 4 (PF4), the role of individual genetic susceptibility remains poorly understood. The aim of this thesis was to investigate the genetic profiles of patients diagnosed with vaccine-induced immune thrombotic thrombocytopenia (VITT) following SARS-CoV-2 adenoviral vaccination. Between February and June 2021, several suspected VITT cases were referred to the Center for Atherothrombotic Diseases at the University of Florence and Careggi University Hospital, and only patients with a possible or definite VITT diagnosis were included in a prospective registry. The cohort therefore included 10 patients (7 females and 3 males), with a median age of 63.5 years [IQR 41–78]. The median interval between vaccination and clinical onset was 8 days [IQR 6–23]. After DNA extraction from peripheral blood, molecular characterization using Whole Exome Sequencing (WES) was performed on six of the ten patients at the Advanced Genetic-Molecular Laboratory. All participants were women of Caucasian origin, with a median age of 64.2 ± 13.8 years. Five had received the VAXZEVRIA (AstraZeneca) vaccine and one the JANSSEN (Johnson & Johnson) vaccine. The study aimed to identify potential genetic variants associated with susceptibility to VITT, providing insights into the molecular mechanisms underlying this rare vaccine-related adverse event. Variants were prioritized based on minor allele frequency (<0.01), functional impact, and pathogenicity predicted using in silico tools (e.g., PolyPhen-2, MutationTaster, PROVEAN). Genes of interest were selected based on their involvement in coagulation, platelet activation, integrin signaling, and thrombotic thrombocytopenia, as defined by Gene Ontology and literature data. Pathogenicity was assessed according to ACMG guidelines. In total, 194 heterozygous variants were identified across the six patients. According to ACMG criteria, 47 of these variants (24.2%) were classified as Variants of Uncertain Significance (VUS), while the remaining ones were considered benign or likely benign. No variant was classified as likely pathogenic or pathogenic. Notably, individual patients harbored rare variants in genes such as STAB2, GP6, SERPINA12, FGA, CD47, and MFSD2B, which are involved in platelet function, coagulation, or vascular signaling. No enrichment of common functional pathways was identified across the cohort, reflecting potential genetic heterogeneity. Some variants were found in genes previously implicated in thrombotic risk or immune regulation, such as ITGA2B, THBD, and VASP. In this thesis we have done whole exome sequencing to investigate the genetic underpinnings of VITT. Although no single causal variant or pathway was identified across patients, the presence of multiple rare variants affecting thrombosis-related genes supports the hypothesis that individual genetic backgrounds may influence susceptibility to VITT. The findings highlight the need for larger, multicenter genomic studies to clarify the genetic contribution to vaccine-related thrombotic events, as well as functional validation of VUS. These insights may eventually contribute to personalized risk assessment for rare vaccine-related adverse events.