Author/Editor		Mitrovič, Mitja; Potočnik, Uroš
Title		High resolution melting curve analysis for high-throughput SNP genotyping in IL23R gene and association of IL23R with Slovenian inflammatory bowel diseases patients
Type		članek
Source		Acta Chim Slov
Vol. and No.		Letnik 57, št. 2
Publication year		2010
Volume		str. 498-505
Language		eng
Abstract		Single nucleotide polymorphism (SNP) analysis is important tool in the studies of genetic factors associated with complex diseases and with genetically influenced response to drug therapy (pharmacogenetics). Recently, a new generation of generic dsDNA binding dyes (LCGreenTM) contributed to the development of fast and low-cost method for SNP detection and/or genotyping based on high resolution melting (HRM) analysis. The aim of our study was to develop HRM assay for IL23R gene (rs7517847) and to perform association study in Slovenian inflammatory bowel diseases (IBD) patients. We genotyped 345 Slovenian healthy controls and 295 IBD patients including 159 with Crohn's disease (CD) and 136 with ulcerative colitis (UC) for rs7517847 polymorphism in IL23R gene using standard RFLP and optimized HRM methods. In this study, we showed, that HRM is a simple, fast and reliable method for genotyping of clinical samples where homozygotes (GG and TT) were determined by Tm calling method and difference between homozygotes and heterozygotes was determined by different melting curve shape using gene scanning method. With combination of results from Tm calling and gene scanning methods, we achieved 98,6% concordance between PCR-RFLP and PCRHRM results, based on the analysis of 640 samples. We found statistically significant association of IL23R polymorphism with Slovenian Crohn's disease patients when comparing genotype and allele frequencies between CD patients and controls. Allele frequency of minor allele G was 0,46 in controls and was reduced to 0,33 in CD patients (p < 0,001, OR = 0,588). The frequency of T/T genotype carriers was higher in CD patients (50,3%) than in controls (26,7%, p = 0,002, OR = 2,558). We found weak association between IL23R polymorphism and Slovenian UC patients. Carriers of T/T genotype have higher risk for UC (p = 0,035, OR = 1,599). (Abs. trunc. at 2000 ch.)
Summary		Analiza polimorfizmov posameznega nukleotida (ang. SNP za single nucleotide polymorphism) in mutacij je ključnega pomena pri ugotavljanju genetskih dejavnikov tveganja za nastanka kompleksnih bolezni in genetsko pogojenega odziva posameznikov na zdravila (farmakogenetika). Razvoj nove generacije fluorescentnih označevalcev (LCGreen), ki se z večjo afiniteto vežejo na dvojno vijačnico DNK, je omogočil razvoj nove metode za hitro in cenovno ugodno analizo polimorfizmov SNP in mutacij na osnovi analize talilne krivulje visoke ločljivosti (ang. HRM za Highresolution melting). Namen naše študije je bil razvoj metode HRM za gensko tipizacijo polimorfizma rs7517847 v genu IL23R in izvedba asociacijske študije za omenjeni polimorfizem pri slovenskih bolnikih s kronično vnetno črevesno boleznijo (KVČB). V asociacijski študiji smo z uporabo standardne metode PCR-RFLP in optimizirane metode PCRHRM določili genotipe za polimorfizem gena IL23R (rs7517847) za 345 zdravih posameznikov in za 295 bolnikov s KVČB, med katerimi je bilo 159 bolnikov s Crohnovo boleznijo (CB) in 136 bolnikov z ulceroznim kolitisom (UK). Ugotovili smo, da je HRM enostavna, hitra in zanesljiva metoda za določanje genotipov v kliničnih vzorcih. Razlike med skupinama homozigotov (GG in TT) smo ugotavljali z algoritmom Tm calling, heterozigote in homozigote pa smo razlikovali na osnovi različnih oblik talilnih krivulj z algoritmom gene scanning. S kombinacijo obeh algoritmov za analizo HRM podatkov smo določili genotipe za 640 vzorcev in ugotovili 98,6% skladnost z genotipi, ki so bili določeni z referenčno RFLP metoda na istih vzorcih. V asociacijski študiji smo s primerjavo alelnih in genotipskih frekvenc med zdravimi posamezniki in bolniki s CB odkrili statistično značilno povezavo med polimorfizmom gena IL23R in skupino bolnikov s CB. Pri zdravih posameznikih je bila alelna frekvenca alela G 0,46, pri bolnikih s CB pa 0,33 (p < 0,001, OR = 0,588). (Izvl. skrajšan na 2000 zn.)
Descriptors		INFLAMMATORY BOWEL DISEASES GENOTYPE MUTATION