doktorska disertacija
Lovro Vidmar (Author), Borut Peterlin (Mentor)

Abstract

Multipla skleroza (MS) je kronična vnetna demielinizacijska in nevrodegenerativna bolezen centralnega živčnega sistema (CŽS). Velja za neozdravljivo bolezen in predstavlja enega izmed poglavitnih vzrokov za invalidnost mladih odraslih v razvitem svetu. Etiologija MS še ni poznana, znano pa je, da gre za multifaktorsko bolezen, ki se razvije kot posledica prispevka dednih dejavnikov, dejavnikov okolja in njihovega medsebojnega vplivanja. Dosedanje raziskave dednih dejavnikov so odkrile številne pogoste genetske različice, ki pa skupaj pojasnijo le manjši delež heritabilitete MS. Možne kandidate za pojasnitev manjkajoče heritabilitete pri multifaktorskih boleznih predstavljajo v populaciji redke genetske različice, katerih preučevanje nam je v zadnjem času omogočila tehnologija sekvenciranja naslednje generacije (NGS). V luči naših preteklih odkritij in ostalih dokazov za pomembnost inflamasoma za MS smo postavili hipotezo, da redke kodirajoče genetske različice vplivajo na funkcionalnost in interakcije proteinov vpletenih v regulacijo inflamasoma, kar pri posamezniku poviša tveganje za MS. MS je znano povezana z izražanjem humanih endogenih retrovirusov (HERV), ki predstavljajo 8 % celotnega človeškega genoma. Nedavno so v znanstveni literaturi objavili več lokusov, na katerih so insercije HERV zaporedij prisotne pri le določenih posameznikih. Te predstavljajo do sedaj neraziskano genetsko raznolikost pri človeku, ki je zanimiva v kontekstu s HERV-povezanimi boleznimi, kot je MS. Med okoljskimi dejavniki tveganja za MS je najvidnejša okužba s herpes virusom 4, poznanim tudi kot virus Epstein-Barr (EBV). Med dokazano pomembne dejavnike pa prištevamo še kajenje. Cilj naše raziskave je predstavljalo iskanje manjkajoče heritabilitete MS in integracija na novo odkritih genomskih dejavnikov tveganja z znanimi ekspozomskimi. V raziskavo smo vključili preiskovance, ki so bili razvrščeni v eno izmed treh skupin: preiskovanci s sporadično obliko MS (skupina MSS), preiskovanci z družinsko oziroma familiarno obliko MS (MSFAM), kontrolni preiskovanci (skupina KONTR). Osnovno metodo v raziskavi je predstavljala metoda sekvenciranja celotnega eksoma (WES), ki temelji na tehnologiji NGS. WES nam je omogočila odkrivanje izjemno redkih različic v panelu 62 genov, ki znano vplivajo na regulacijo inflamasoma, ter genotipizacijo HLA lokusov DRB1*15:01 in HLA-A*02:01 pri 319 preiskovancih. Pri primerjavi mutacijskega bremena med skupinami preiskovancev smo upoštevali obtežitev različic na podlagi njihove frekvence v podatkovni bazi “gnomAD” ter njihove ocene z algoritmom za napovedovanje njihove funkcijske pomembnosti CADD. V prvi fazi smo na 102 preiskovancih z v ta namen razvito ciljano NGS metodo sistematično določili frekvenco HERV insercij na 20 znano polimorfnih lokusih in za dva, ki sta dosegla značilno obogatitev med bolniki z MS, preiskavo razširili na 253 preiskovancev z uporabo PCR dolgega dosega. Zanje smo z metodo realno-časovne kvantitativne PCR določili tudi prisotnost oziroma število kopij genoma EBV v DNA izolirane iz celotne periferne krvi preiskovancev. Epigenetske analize stopnje metilacije znano s kajenjem povezanih CpG lokusov smo opravili na 102 preiskovancih. Glavne rezultate raziskave predstavljajo: Odkritje statistično značilno povišanega mutacijskega bremena redkih funkcijskih različic v inflamasomski poti pri preiskovancih z MS, pri čemer se breme med skupinama MSS in MSFAM ni razlikovalo. S sistematično genotipizacijo v populaciji nefiksiranih insercij endogenih retrovirusov smo identificirali inserciji v genih PTPRN2 in RASGRF2 kot dedna dejavnika tveganja za MS, pri čemer je bila povišana prisotnost insercije v RASGRF2 značilna le za skupino MSFAM. Na preiskovancih vključenih v raziskavo smo potrdili povezanost alela HLA-DRB1*15:01 z MS in odkrili njegov sinergični učinek z odsotnostjo protektivnega alela HLA-A*02:01. V krvi bolnikov z družinsko obliko MS smo v primerjavi s kontrolnimi preiskovanci statistično značilno pogosteje odkrili virus Epstein-Barr in v isti skupini preiskovancev dokazali povišano število kopij virusa v krvi. Povezav med preiskovanimi genomskimi dejavniki tveganja in EBV viremijo nismo zaznali. Vsak preiskovani dejavnik je ob njegovi vključitvi v model zgrajen z učnimi drevesi izboljšal njegovo napovedno vrednost statusa MS. Na podlagi pregleda znanstvene literature smo prikazali učinek kajenja na vsakega izmed preučevanih dejavnikov tveganja in vse najvidnejše okoljske in genomske dejavnike tveganja za MS umestili v inflamasomko regulatorno pot. Na podlagi dokazane povezanosti inflamasoma z vsemi preučevanimi dejavniki, predlagamo inflamasom kot osrednje stičišče posameznih dejavnikov tveganja za MS in izpostavljamo njegov pomen za etiopatogenezo MS.

Keywords

multipla skleroza;sekvenciranje nove generacije;inflamasom;mutacijsko breme;redke genetske različice;humani endogeni retrovirusi;HERV;kajenje;EpsteinBarr;molekularna genetika;ekspozom;disertacije;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL BF - Biotechnical Faculty
Publisher: [L. Vidmar]
UDC: 575:616.8(043.3)=163.6
COBISS: 41241347 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Integrative analysis of selected genomic and exposomic factors in multiple sclerosis
Secondary abstract: Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the central nervous system (CNS). To date there is no effective treatment and, due to its debilitating physical and cognitive symptoms, MS is the most common cause for disability of young adults in the developed world. While the etiology of MS is yet to be elucidated, it is known to be a multifactorial disease, which develops as a consequence of genetic and environmental risk factors and their interaction. GWA studies performed to date have discovered numerous common genetic variants, which collectively do not explain the observed heritability of the disease. One plausible candidate for the explanation of missing heritability in multifactorial diseases are exceedingly rare genetic variants whose detection is now enabled by the next-generation sequencing technology (NGS). In the light of our previous research results and existing evidence linking inflammasome to MS, we investigated the mutational burden in the genes comprising the inflammasome regulatory network. MS has been associated with increased transcription of human endogenous retroviruses (HERV) which constitute around 8 % of the human genome. Recently, several genomic loci with populationally unfixed HERV insertions were published. These represent an uncharacterized source of human genetic variability, which is interesting in the context of HERV associated diseases, such as MS. The most prominent environmental MS risk factors are infection with Epstein-Barr virus (EBV) and smoking. The aim of our research was the search for missing heritability of MS and the integration of newly found genetic risk factors with the known environmental ones. Probands included in the research belonged to either of the three groups: Probands with sporadic MS (MSS), probands from families with multiple affected members (MSFAM), and ethnically matched control group. We employed whole exome-sequencing (WES) which is based on NGS technology as our main experimental method. The method enabled us to genotype all genetic variants in the panel of 62 genes which are known to be associated with the regulation of NLRP1/NLRP3 inflammasome. Additionally, we used bioinformatics tools to obtain the genotypes of the main risk and protective HLA alleles for MS: DRB1*15:01 and HLA-A*02:01. These two methods were applied to all 319 probands. Mutational burden analysis was weighted with populational variant frequency obtained from the “gnomAD” database and with the results of variant pathogenicity prediction algorithm: CADD. In the first phase of the research, we employed and additional method, a custom-designed targeted NGS, to systematically genotype 20 populationally unfixed HERV insertions on 102 probands. Two HERV insertions (located in the introns of RASGRF2 and PTPRN2 genes) were enriched amongst the MS probands and were genotyped on additional probands (total of 253) using the long-range PCR, which confirmed their significant association with MS. For the same set of 253 probands, we performed a quantitative estimation of EBV viremia using the real-time qPCR method. Epigenetic analysis of differential methylation of CpG loci known to be associated with smoking status was performed on 102 probands. We have discovered a significantly increased rare variant burden in the inflammasome regulatory genes of patients with MS, which was most prominent for the exceedingly rare variants that were scored highly by the pathogenicity prediction algorithm. These variants support the overall importance of inflammasome and its regulation by interferon-β and auto/mitophagy to the etiology of MS. In the present study, we report insertion allele frequencies for 20 unfixed HERV-K insertions and demonstrate two of them to be overrepresented among the patients with MS, compared to the control cohort. The HERV-K sequence within the RASGRF2 gene was detected only in the heterozygous state and was overrepresented only in the MS patients from families with multiple affected members. The HERV-K sequence inserted within the PTPRN2 gene was overrepresented in both familial and sporadic MS patients, in both heterozygous and homozygous state, and was associated with MS according to the log-additive model applied. Both insertions were present as solo LTRs located within the intronic sequences of the respected genes. We describe an additive synergic effect between the presence of DRB1*15:01 and the absence of HLA-A*02:01 alleles in the investigated population. The analysis of EBV viremia showed a significant increase of EBV presence in the blood of MSFAM probands, however, the one amongst MSS was similar to the control group. Both MSS and the control group displayed a trend in which EBV viremia increased with probands' age at blood draw. We did not find any significant correlation between the level of EBV viremia and the investigated genomic risk factors. In the joint analysis of all measured MS risk factors, each of them contributed to the accuracy of the decision tree models. Based on the documented interactions of the NLRP1/NLRP3 inflammasome pathway with other MS risk factors and interferon-β afferent and efferent pathways, we propose inflammasome as the central integrator of genomic and environmental risk factors for MS and put forward its importance for MS etiology.
Secondary keywords: multiple sclerosis;next generation sequencing;inflammasome;mutational burden;rare genetic variants;human endogenous retroviruses;HERV;smoking;Epstein-Barr;molecular genetics;exposome;dissertations;
Type (COBISS): Doctoral dissertation
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak.
Pages: X, 100 f.
ID: 12152199