Transkriptomski profil med multisistemskim vnetnim sindromom pri otrocih

Križnar, Pia (author), Trebušak Podkrajšek, Katarina (mentor), DEBELJAK, MARUŠA (co-mentor)

Multisistemski vnetni sindrom pri otrocih (MIS-C) je nov, imunsko posredovan sindrom. Gre za življenjsko nevaren, hipervnetni sindrom. Običajno za MIS-C zbolijo otroci, ki so bili predhodno zdravi in niso imeli hudega poteka bolezni COVID-19, po dveh do šestih tednih po preboleli okužbi s SARS-CoV-2 pa nastopi visoka vročina, močno vnetje in znaki večorganske okvare. Podatkov o kliničnem poteku bolezni in prognozi je malo. Pojavnost MIS-C je bila ocenjena na približno 1 do 10 oseb na 1 000 000 otrok in mladostnikov do 21 let. Pri iskanju genetskih dejavnikov tveganja za MIS-C je ena od strategij prepoznavanje diferencialnega izražanja genov s sekvenciranjem RNA. S tem so bili prepoznani geni, ki imajo izrazito večje izražanje pri bolnikih z MIS-C kot pri zdravih kontrolah in sodelujejo v bioloških procesih, kot so prirojen in pridobljen imunski odziv, odziv na virus, signalne poti posredovane s citokini, signalna pot interferona tipa I, II, sistem komplementa in signaliziranje NF-κB. Namen magistrske naloge je preučiti razlike v izražanju genov pri bolnikih v aktivni fazi bolezni MIS-C in pri bolnikih v remisiji, ki so služili kot kontrolna skupina, z metodo NGS in nadaljnjo bioinformatsko analizo, ter prepoznati patofiziološke procese, v katere so ti geni vpleteni. Izolirali smo RNA iz polne krvi bolnikov v času aktivne faze bolezni in v času remisije, nato smo pripravili knjižnice RNA in izvedli sekvenciranje z aparatom NovaSeq 6000, Illumina. Z uporabo podatkovnega cevovoda Snakepipes DeSeq2 in spletnega orodja Enrichr smo preučili diferencialno izražanje genov in poti, v katerih ti geni sodelujejo. Z uporabljenim pristopom smo uspeli ločiti med preiskovanci v aktivni fazi bolezni in med tistimi v remisiji. Ločitev bi lahko izboljšali z optimizacijo bioinformatske analize. Pri bolnikih z MIS-C smo ugotovili povečano aktivacijo imunsko povezanih poti, vključno z aktivacijo nevtrofilcev, uravnavanjem citokinov in interferonov, kaskado TLR, procesiranjem in predstavitvijo antigenov ter signaliziranjem NF-κB. Med drugim smo odkrili tudi obogatene poti označevalcev vnetja, humoralnega imunskega odziva, aktivacije komplementa in koagulacijskih poti. Bolniki z MIS-C so pokazali izrazito inhibicijo signalnih poti T-celičnega receptorja in signalnih poti, povezanih z limfociti T. Potrebne pa so nadaljnje raziskave, ki bodo natančneje opredelile njihov pomen v patofiziologiji MIS-C.

• Language: Slovenian
• Keywords: MIS-C, NGS, sekvenciranje RNA, imunski odziv, transkriptomski profil
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-154564
• Publication date in RUL: 21.02.2024

Secondary language

• Language: English
• Title: Transcriptomic signature during multisystem inflammatory syndrome in children

Abstract

Multisystem inflammatory syndrome in children (MIS-C) is a rare, severe complication of COVID-19. It is characterized by non-specific symptoms such as fever, severe inflammation and can lead to severe complications including cardiovascular shock and multi-organ failure. Typically, MIS-C appears in previously healthy children and adolescents about 2-6 weeks after SARS-CoV-2 acute infection. Limited data is available regarding the disease's clinical progression and prognosis. The incidence of MIS-C has been estimated to be approximately 1 to 10 cases per 1,000,000 people under 21 years of age. In the search for genetic risk factors in MIS-C patients, RNA sequencing analysis has identified differentially expressed genes involved in key biological processes such as innate and acquired immune response, virus response, cytokine-mediated signalling pathways, type I and II interferon signalling pathway, complement system and NF-κB signalling. The purpose of this master degree thesis was to explore differential gene expression between patients with confirmed MIS-C and the control group using the NGS method and to examine in which pathophysiological processes the differentially expressed genes are involved. RNA was isolated from patients during the active phase of the disease and during remission. Then RNA libraries were prepared and sequenced using the NovaSeq 6000, Illumina. Using the Snakepipes DeSeq2 pipeline and Enrichr web tool, we examined the differential gene expression and pathways in which these genes are involved. The study successfully distinguished subjects in active phase and controls, with potential for improvement through optimisation of the bioinformatics analysis. MIS-C patients exhibited increased activation of immune-related pathways, including neutrophil activation, cytokine and interferon regulation, TLR cascade, antigen processing and presentation, and NF-κB signalling. Among others, we also discovered enriched pathways related to markers of inflammation, humoral immune response, complement activation and coagulation pathways. Patients with MIS-C showed marked inhibition of T-cell receptor and T-lymphocyte-related signalling pathways. Further studies are needed to fully elucidate their role in pathophysiology of MIS-C.

• Keywords: MIS-C, NGS, RNA-sequencing, immune response, transcriptomic signature