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Title:High resolution melting curve analysis for high-throughput SNP genotyping in IL23R gene and association of IL23R with Slovenian inflammatory bowel diseases patients
Authors:ID Mitrovič, Mitja (Author)
ID Potočnik, Uroš (Author)
Files:.pdf Acta_Chimica_Slovenica_2010_Mitrovic,_Potocnik_High_Resolution_Melting_Curve_Analysis_for_High-Throughput_SNP_Genotyping_in_IL23R_Gene_a.pdf (232,19 KB)
MD5: 7012F6819B1DE9B1AD10E85A7B766FBA
PID: 20.500.12556/dkum/c5f8ee3f-dea5-4bd6-bfa5-62d08d608b61
 
URL http://acta-arhiv.chem-soc.si/57/57-2-498.pdf
 
Language:English
Work type:Scientific work
Typology:1.01 - Original Scientific Article
Organization:MF - Faculty of Medicine
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). These results suggest IL23R plays important role in CD and UC development in Slovenian patients.
Keywords:SNP genotyping, high resolution melting, DNA dyes, inflammatory bowel diseases, LC Green Plus
Publication status:Published
Publication version:Version of Record
Year of publishing:2010
Number of pages:str. 498-505
Numbering:Letn. 57, št. 2
PID:20.500.12556/DKUM-67455 New window
ISSN:1318-0207
UDC:616.34-002
ISSN on article:1318-0207
COBISS.SI-ID:3649343 New window
NUK URN:URN:SI:UM:DK:RJNTFVG7
Publication date in DKUM:18.08.2017
Views:1939
Downloads:118
Metadata:XML DC-XML DC-RDF
Categories:Misc.
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MITROVIČ, Mitja and POTOČNIK, Uroš, 2010, High resolution melting curve analysis for high-throughput SNP genotyping in IL23R gene and association of IL23R with Slovenian inflammatory bowel diseases patients. Acta Chimica Slovenica [online]. 2010. Vol. 57, no. 2, p. 498–505. [Accessed 27 April 2025]. Retrieved from: https://dk.um.si/IzpisGradiva.php?lang=eng&id=67455
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Record is a part of a journal

Title:Acta Chimica Slovenica
Shortened title:Acta Chim. Slov.
Publisher:Slovensko kemijsko društvo
ISSN:1318-0207
COBISS.SI-ID:14086149 New window

Licences

License:CC BY 4.0, Creative Commons Attribution 4.0 International
Link:http://creativecommons.org/licenses/by/4.0/
Description:This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.
Licensing start date:18.08.2017

Secondary language

Language:Slovenian
Abstract: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). Frekvenca posameznikov z genotipom T/T je bila pri bolnikih s CB (50,3%) višja kot pri skupini zdravih posameznikov (26,7%, p = 0,002, OR = 2,558). Odkrili smo tudi šibko povezavo med polimorfizmom gena IL23R in bolniki z UC, kjer so imeli nosilci genotipa T/T višje tveganje za UC (p = 0,035, OR = 1,599). Ti rezultati kažejo, da igra gen IL23R pomembno vlogo v patogenezi pri slovenskih bolnikih s CB in UC.
Keywords:fluorescentni označevalci, DNK, dvojna vijačnica, talilna krivulja


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