VLOGA DNA POLIMORFIZMA POSAMEZNEGA NUKLEOTIDA rs2476601 IN IZRAŽANJE GENA PTPN22 PRI NASTANKU, POTEKU IN ZDRAVLJENJU OTROŠKE ASTME

Čeh, Alja

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Naslov:	VLOGA DNA POLIMORFIZMA POSAMEZNEGA NUKLEOTIDA rs2476601 IN IZRAŽANJE GENA PTPN22 PRI NASTANKU, POTEKU IN ZDRAVLJENJU OTROŠKE ASTME
Avtorji:	ID Čeh, Alja (Avtor) ID Potočnik, Uroš (Mentor) Več o mentorju... ID Berce, Vojko (Komentor)
Datoteke:	UNI_Ceh_Alja_2014.pdf (1,45 MB) MD5: 9FDAC8F9BAD8820A43C58324902104D9
Jezik:	Slovenski jezik
Vrsta gradiva:	Diplomsko delo
Tipologija:	2.11 - Diplomsko delo
Organizacija:	FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Opis:	Astma je resna kronična bolezen dihal neznane etiologije in je pogostejša pri otrocih kot odraslih. Je kompleksna bolezen s heterogenim fenotipom, na njen pojav vpliva interakcija več različnih genov, pri čemer je prispevek posameznega gena le majhen. Proces interakcije je odvisen od dveh faktorjev, genetike bolnika ter izpostavljenost okolju v času razvoja imunskega sistema. Pri zdravljenju astme prihaja do velikih razlik v odzivu na terapijo, katere so posledica genskega zapisa bolnika. Pri zdravljenju astme se uporabljata dve vrsti zdravil, preprečevalci astme (glukokortikoidi in antilevkotrieni) in olajševalci astme (bronhodilatatorji). V nalogi smo se osredotočili na vlogo gena PTPN22 in polimorfizma posameznega nukleotida (SNP) rs2476601 na genu PTPN22 pri nastanku, poteku in zdravljenju otroške astme. SNP rs2476601 se nahaja na genu PTPN22 in je povezan z nastankom številnih sorodnih avtoimunskih bolezni, kot so Chronova bolezen, revmatoidni artritis, diabetis tipa 1 in multipla skleroza, katere imajo podoben potek bolezni kot astma. Zato je PTPN22 dober kandidatni gen za astmo. Namen diplomske naloge je bil narediti asociacijsko analizo SNP-ja rs2476601 pri slovenskih otrocih z atopijsko in neatopijsko astmo, izvesti farmakogenetsko analizo, primerjati izražanje gena PTPN22 med zdravimi posamezniki in bolnimi posamezniki z atopijskim in neatopijskim fenotipom astme ter primerjati izražanje gena PTPN22 pred in po terapiji. Izbran SNP rs2476601 na genu PTPN22 smo genotipizirali ter naredili primerjavo frekvence genotipov med zdravimi kontrolami in bolniki z astmo, atopijskim in neatopijskim fenotipom ter analizo vpliva genotipa izbranega SNP-ja na klinične in laboratorijske parametre. Izmerili smo razlike v izražanju gena PTPN22 glede na genotip, primerjali razlike v izražanju med zdravimi kontrolami in bolniki z astmo z atopijskim in neatopijskim fenotipom ter spremembo v izražanju po zdravljenju z inhalacijskimi kortikosteroidi, antilevkotrienom in antilevkotrienom intermitentno. Genotipe smo določili z metodo HRM in nekatere preverili z metodo RFLP na DNA, izoliranih iz krvnih limfocitov 276 zdravil kontrol ter 325 bolnih otrok starih med 5 in 18 let, od tega jih je imelo 189 atopijski fenotip in 84 neatopijski fenotip astme. Statistično analizo smo izvedli s programskim paketom SPSS. Za analizo frekvence alelov in primerjavo genotipov bolnih otrok s kontrolami smo uporabili Fisherjev natančni test. Za vpliv genotipov na klinične in laboratorijske parametre smo uporabili T-test, Mann-Whitney test in test ANOVA. Izražanje gena PTPN22 smo izmerili s kvantitativnim PCR v realnem času (qPCR) v krvnih limfocitih 295 bolnih otrok starih med 5 in 18 let, od tega 177 z atopijsko in 44 z neatopijsko astmo, ter v krvnih limfocitih 158 zdravih kontrol. Za ekspresijsko analizo smo imeli na voljo 127 vzorcev po terapiji, od tega 53 zdravljenih z inhalacijskimi kortikosteroidi, 31 z antilevkotrienskim zdravilom Singular in 43 antilevkotrienskim zdravilom Singular intermitentrno. Vpliv genotipa SNP-ja rs2476601 na izražanje gena PTPN22 smo preverili s testom ANOVA in Mann-Whitney testom, primerjavo izražanja gena med bolnimi otroci in zdravimi kontrolami prav tako z Mann-Whitney testom. Vpliv zdravljenja na izražanje gena smo preverili z Wilcoxonovim testom. Vse p vrednosti, ki so bile nižje od 0,05, smo upoštevali, kot statistično signifikantne. Ugotovili smo, da je SNP rs2476601 povezan s pojavom astme (p=0,034; OR=0,623) pri slovenskih otrocih. Prav tako smo ugotovili, da se astmatiki z genotipom AG in GG boljše odzivajo na zdravljenje (vse terapije) v primerjavi s posamezniki z genotipom AA, saj se je vrednost FEV1 po terapiji pri teh posameznikih povečala (p=0,033; +/-s.d=13,273). Za skupino astmatikov z atopijskim fenotipom smo dobili signifikantne rezultate za vrednost eozinofilcev, pri čemer smo za posameznike z genotipom AA ugotovili višjo vrednost v primerjavi s posamezniki z genotipom AG in GG, kar kaže na to, da imajo posamezniki z genotipom AA večjo nagnjenost k atopiji.
Ključne besede:	astma, rs2476601, PTPN22, farmakogenomika, HRM, RFLP, qPCR
Kraj izida:	Maribor
Založnik:	[A. Čeh]
Leto izida:	2014
PID:	20.500.12556/DKUM-46784
UDK:	575.111:616.248(043.2)
COBISS.SI-ID:	18398998
NUK URN:	URN:SI:UM:DK:6YMVAW0Q
Datum objave v DKUM:	26.11.2014
Število ogledov:	2709
Število prenosov:	193
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Sekundarni jezik

Jezik:	Angleški jezik
Naslov:	THE ROLE OF SINGLE NUCLEOTIDE POLYMORPHISM rs2476601 AND PTPN22 GENE EXPRESSION IN DEVELOPMENT, PROGRESSION AND TREATMENT OF CHILDHOOD ASTHMA
Opis:	Asthma is a serious chronic inflammatory disease of the airways of unknown etiology and it is more common with children than with adults. Asthma is a complex disease with a heterogeneous phenotype. Its occurrence may be influenced by the interaction of various genes. The process of the interaction depends on two factors, namely on the patient's genetics and on the environment to which the patient was exposed during the development of the immune system. During the treatment, there are noticeable differences in the reaction to therapy, which result from the patient's genome. Asthma is treated by two kinds of medications: prevention medications (glucocorticosteroids and leukotriene antagonists) and relief medications (bronchodilators). The work focuses on the role of the single nucleotide polymorphism (SNP) rs2476601 and the expression of gene PTPN22 at the occurrence, process and treatment of childhood asthma. SNP rs2476601, which is located in the PTPN22 gene, is associated with a number of other autoimmune diseases such as Crohn's disease, rheumatoid arthritis, diabetis type 1 and multiple sclerosis, which are similar to the course of the disease, such as asthma. Therefore, PTPN22 can be a good candidate gene for asthma. The purpose of the work is to perform association analysis of SNP rs2476601 in Slovene children suffering from atopic and non-atopic asthma. Secondly, the goal of the work is to produce the pharmacogenetic analysis, to compare the expression of gene PTPN22 of healthy controls with patients with atopic and non-atopic phenotype and finally, to compare the expression of gene PTPN22 before and after the therapy. The chosen SNP rs2476601 on gene PTPN22 was genotyped. We compared frequency of genotypes between healthy controls and patients suffering from asthma, both atopic and non-atopic. Furthermore, an analysis was made which focused on the impact of the genotypes for SNP rs2476601 on clinical and laboratory parameters. We analyzed expression of gene PTPN22 according to the genotype of SNP rs2476601, the differences in the expression between healthy controls and patients suffering from atopic and non-atopic asthma. Genotyping was performed by HRM and RFLP methods on DNA isolated from blood lymphocytes of 276 healthy controls and 325 children with asthma, aged 5 – 18, out of which 189 had the atopic phenotype and 84 had the non-atopic phenotype. The statistical analysis was performed with the SPSS software. To compare allele and genotype frequencies between asthmatic children and controls we used Fisher exact test. To examine the impact of genotypes on clinical and laboratory parameters we used T-test, Mann-Whitney test and ANOVA test. The expression of gene PTPN22 was measured using real time PCR (qPCR) in blood lymphocytes of 295 asthmatic children aged 5 – 18, out of which 177 with atopic asthma and 44 had non-atopic asthma and 158 healthy controls. There were 127 samples after therapy available, of which 53 were treated with inhaled corticosteroids, 31 with leukotriene antagonist Singular and 43 with leukotriene antagonist Singular, intermittently. The impact of genotypes on gene expression was analyzed with ANOVA test and Mann-Whitney test. The comparison of the gene expression between asthmatic children and healthy controls was made with Mann-Whitney test as well. The impact of treatment on gene expression was performed with Wilcox test. All p values, lower than 0,05, were regarded as statistically significant. The results confirmed our hypothesis that SNP rs2476601 is associated with the occurrence of asthma in Slovenian children (p=0,034; OR=0,623). We have also found that asthmatics with AG and GG genotype had better response to treatment (all treatments) compared to individuals with the AA genotype, as the value of FEV1 after therapy has increased in individuals with AG and GG genotype (p = 0,033; +/- s.d = 13,273). For a group of asthmatics with atopic phenotype, we found significant association with the number of eosinophils, where individuals with AA genotype had higher value compare
Ključne besede:	asthma, rs2476601, PTPN22, pharmacogenomics, HRM, RFLP, qPCR

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