20.500.12556/DKUM-62229 eQTL ANALIZA KROMOSOMSKIH REGIJ 17q21 IN 2q12 TER NJUN VPLIV NA RAZVOJ IN POTEK ASTME PRI SLOVENSKIH OTROCIH eQTL ANALYSIS OF CHROMOSOMAL REGION 17q21 AND 2q12 THEIR ROLE IN DEVELOPMENT AND PROGRESSION OF CHILDHOOD ASTHMA IN SLOVENIAN CHILDREN Astma je resna kronična bolezen pljuč, ki povzroča vnetje dihal in je neznane etiologije. Sam razvoj astme je povezan z interakcijo številnih genov in vplivom okolja. V številnih študijah so z astmo povezali več kot 100 genov, ki pa se med različnimi populacijami razlikujejo. Raziskali smo dve kandidatni kromosomski regiji, 17q21 in 2q12. Za analizo 17q21 smo genotipizirali polimorfizem posameznega nukleotida (SNP) rs7216389, ter merili izražanje genov ORMDL3 in GSDMB, za analizo regije 2q12 pa smo genotipizirali SNP rs10206753 in merili izražanje genov IL1RL1 in IL18RAP. S tehniko verižne reakcije s polimerazo (PCR), ki ji je sledila reakcija polimorfizmov dolžin restrikcijskih encimov (RFLP) ali analiza talilne krivulje visoke ločljivosti (HRM), smo genotipizirali 384 vzorcev otrok z astmo z atopijskim in neatopijskim fenotipom ter 288 vzorcev zdravih kontrol. Analizirali smo povezavo izbranih SNP-jev z astmo ter vpliv izbranih SNP-jev na klinične parametre in izražanje genov ORMDL3, GSDMB, IL1RL1, ter IL18RAP. Ugotovili smo, da je SNP rs7216389 povezan s pojavom astme pri slovenskih otrocih (p=0,0031, OR=0,698). Astmatiki, atopijski in neatopijski, imajo višjo frekvenco alela T v primerjavi z zdravimi kontrolami (p=0,0005, OR=0,541). Farmakogenetska analiza SNP-ja rs7216389 je pokazala, da se atopijski in neatopijski astmatiki različno odzivajo na terapijo z inhalacijskimi glukokortikosteroidi. Atopijskim astmatikom z genotipom CC se vrednost dTI po terapiji poviša (p=0,0057, dTICC=7,00 , dTICT+CC=2,00), medtem ko se neatopijskim astmatikom zniža (p=0,0311, dTICC=-2,00 ; dTICT+CC=3,00). Pri vplivu genotipa na klinične parametre smo opazili tudi višjo vrednost eozinofilcev v krvi neatopijskih astmatikov z genotipom TT v primerjavi s posamezniki z genotipom CT ali CC, vendar je bila vrednost p na meji statistične značilnosti (p=0,0558). Ugotovili smo tudi, da SNP rs7216389 regulira izražanje gena ORMDL3, saj imajo astmatiki in atopijski astmatiki z genotipom TT višji nivo izražanja gena ORMDL3 v primerjavi s posamezniki z genotipom CT ali TT (p=0,0038 za astmatike, p=0,0133 za atopijske astmatike). Za območje 2q12 pa smo ugotovili, da SNP rs10206753 ni povezan s pojavom astme pri slovenskih otrocih. Smo pa ugotovili, da se vrednost dFEV1 po terapiji z antilevkotrieni v primerjavi s posamezniki z genotipom CT ali TT zviša pri astmatikih z genotipom CC (p=0,0002, 8,5/-2) in pri atopijskih astmatikih z genotipom CC (p=<0,0001; 11/-2). Pri primerjavi izražanja gena IL18RAP med astmatiki in zdravimi kontrolami smo ugotovili, da imajo astmatiki, tako atopijski kot neatopijski, nižje izražanje gena v primerjavi z zdravimi kontrolami (p<0,0001). Rezultati nakazujejo na pomembno vlogo obeh regij, 17q21 in 2q12, tako v patogenezi kot tudi farmakogenetiki astme pri slovenskih otrocih. V prihodnosti bi bilo smiselno opraviti farmakogenetske študije za oba lokusa tudi pri drugih populacijah, s čemer bi naredili korak naprej k personalizirani medicini. Asthma is a serious chronic lung disease that causes inflammination of airways and is of unknown etiology. The development of asthma is related to the interaction of numerous genes and the environment. Numerous studies have linked asthma with more than 100 genes. These candidate genes vary among different populations. We investigated two candidate chromosomal regions, 17q21 and 2q12. For analysis of 17q21 we genotyped single nucleotid polymorphism (SNP) rs7216389, and mesuered expression of ORMDL3 and GSDMB genes. For analysis of region 2q12 we genotyped SNP rs10206753 and mesuered the expression of IL1RL1 and IL18RAP genes. With the method of polymerase chain reaction (PCR), folowed by restriction fragment lenght polymorphism (RFLP) or high resolution melting curve analysis (HRM) we genotyped 384 samples of children with asthma and 288 samples of healthy controls. We analyzed association between selected SNPs, asthma, clinical parameters and expression of ORMDL3, GSDMB, IL1RL1 and IL18RAP genes. We found, that SNP rs7216389 is asociated with the onset of asthma (p=0,0031; OR=0,698) in Slovenian children. Asthmatics, atopic asthmatics and nonatopic asthmatics have higher frequency of T allele compered to healthy controls (p=0,0005 ; OR=0,541). The pharmacogenetic analysis of SNP rs7216389 showed that atopic and nonatopic asthmatics react differently to therapy with inhalation glucocorticosteroids. Atopic asthmatics with genotype CC have better response, higher number of dTI after therapy (p=0,0057 ; dTICC=7,00 , dTICT+CC=2,00), while nonatopic asthmatics with genotype CC have a negative response, lower number of dTI after therapy (p=0,0311 ; dTICC=-2,00 ; dTICT+CC=3,00). When comparing genotypes according to clinical parameters, we found that nonatopic asthmatics with genotype TT have higher number of blood eosinophils than individuals with CT or CC genotype, but the p value was on the border of statistical significance (p=0,0558). We also found that SNP rs7216389 regulates the expression of ORMDL3 gene; asthmatics and atopic asthmatics with genotype TT have higher level of ORMDL3 expression than individuals with CT or TT genotype (p=0,0038 for asthmatics, p=0,0133 for atopic asthmatics). 2q12 locus, we showed that SNP rs10206753 is not associated with the onset of asthma in Slovenian children. However, we found, that dFEV1 is higher in all asthmatics and atopic asthmatics with CC genotype compared to asthmatics with CT or TT genotype, after therapy with antileucotriens (p=<0,0001 ; 11/-2). The association analysis of expression levels of IL18RAP gene beetwen asthmatics and healthy controls showed that asthmatics, atopic and nonatopic asthmatics have lower expressions levels of IL18RAP gene in comparison with healthy controls (p=<0,0001; 0,4/0,42/0,33/1,0). Our results showed that loci 17q21 and 2q12 have an important role in pathogenesis as well as in pharmacogenetics of asthma in Slovenian children. Further studies of both loci in different populations are required to make a step forward in medicine personalized medicine. astma eQTL farmakogenetika ORMDL3 IL1RL1 asthma eQTL pharmacogenetics ORMDL3 IL1RL1 true true false [L. Maksimović] Slovenski jezik Angleški jezik Diplomsko delo 2016-08-24 12:24:21 2016-10-04 14:04:23 2022-06-28 03:08:33 2019-07-11 12:48:17 2016 0 Maribor 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 616.248-053.2(043.2) 20299286 URN:SI:UM:DK:IUL3EPBG UN_Maksimovic_Laura_2016.pdf UN_Maksimovic_Laura_2016.pdf 1 45C2F67B5EB221EBA7B9DA2EB715A866 8e529f9ccca9aa55df450e382feff92f1a009690f1dc67ec489bc8bf05dcf7e8 8e1f68f5-7c0d-11eb-bb7a-00155d0001ca https://dk.um.si/Dokument.php?lang=slv&id=105434 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0