POLIMORPHYSMS IN SELECTED APOPTOSE GENES AS PREDICTIVE BIOMARKERS FOR RESPONSE TO ADALIMUMAB TREATMENT IN CROHN DISEASE PATIENTS

Kronična vnetna črevesna bolezen (KVČB) je kompleksna bolezen, ki se lahko pojavi predvsem kot Crohnova bolezen (CB) ali kot ulcerozni kolitis (UK). Na razvoj bolezni vplivajo tako okoljski kot tudi genetski dejavniki. Bolniki, ki so oboleli za KVČB, zelo različno odgovarjajo na različna zdravila, zato zdravljenje poteka v več stopnjah. Najprej se preizkusijo 5-aminosalicati, nato kortikosteroidi in nazadnje imunosupresivi. Pri Crohnovih bolnikih, ki so neodzivni na omenjena standardna zdravila, se v zadnjem času uporabljajo nove biološke učinkovine, kot sta infliximab (komercialno dostopno zdravilo pod imenom Remicade) in najnovejše zdravilo adalimumab (komercialno dostopno zdravilo pod imenom Humira), ki inhibirajo provnetni citokin TNF-ï¡. Namen dela je bil poiskati farmakogenomske biooznačevalce, ki bi potencialno služili kot napovedni dejavniki odziva na biološko učinkovino adalimumab pri bolnikih s Crohnovo boleznijo. V nalogi smo se osredotočili na analizo polimorfizmov v izbranih apoptoznih genih TIMP-1 (SNP +372C/T oz. rs4898), CASP-9 (SNP 93C/T oz. rs4645983), FCGR3A (SNP 158V/V oz. rs396991 in FAS (SNP -670C/T oz. rs1800682), ki so predhodno pokazali povezavo z odzivom na infliximab. Namen je bil tudi ugotoviti ali so ti polimorfizmi v apoptoznih genih povezani tudi s tveganjem pri slovenskih bolnikih s Crohnovo boleznijo, ki ne odgovarjajo na standardno terapijo oziroma je bila standardna terapija prekinjena zaradi prehudih stranskih učinkov. Prav tako smo želeli raziskati ali obstaja in kakšna je korelacija med polimorfizmi v zgoraj navedenih genih in kliničnimi značilnostmi bolnikov. Vzorci krvi za izolacijo DNK ter za določitev laboratorijske krvne slike in drugih parametrov so bili odvzeti pred prvim odmerkom zdravila in nato še po 4., 12., 20. in 30. tednih. Pri vsakem odvzemu se je stanje bolezni analiziralo še z indeksom IBDQ. Genetske analize izbranih polimorfizmov smo izvedli z metodo RFLP in analizo fragmentov na agaroznem gelu ter s KASPar tehniko. Ugotovili smo povezavo polimorfizma v genu CASP-9 z odzivom na adalimumab. Bolniki z genotipom C/C so imeli slabši odziv opažen predvsem po štirih (p = 0,05) in 20 tednih (p = 0,031) zdravljenja kot bolniki z genotipom C/T ali T/T. Rezultat naše študije je skladen s študijo pri zdravljenju z infliximabom, kjer so bolniki s C/C genotipom v genu CASP-9 prav tako imeli slabši odziv. Pri ostalih 3 analiziranih genih povezava ni bila statisično značilna. Nadalje smo ugotovili povezavo polimorfizmov v genih CASP-9 (SNP rs4645983) in FCGR3A (SNP rs396991) pri slovenskih CB bolnikih vključenih v študijo. Frekvenca alela C na SNP-u rs4645983 gena CASP-9 je pri bolnikih s CB signifikantno nižja (0,68) v primerjavi s frekvenco kontrolne skupine (0,86, p = 9,9*10-5). Prav tako je frekvenca genotipa C/C nižja pri bolnikih s CB (60,22%) v primerjavi z zdravimi posamezniki (78,41%, p = 0,01). Pri primerjavi alelne frekvence SNP-a rs396991 na genu FCGR3A smo ugotovili, da je frekvenca alela T signifikantno nižja pri bolnikih s CB (0,54) v primerjavi z zdravimi posamezniki (0,67, p = 0,01), prav tako se signifikantno razlikuje frekvenca genotipa in sicer 33,3% genotipa T/T pri CB bolnikih v primerjavi s 45,06% pri kontrolni skupini (p = 0.010). Ugotovili smo povezavo med polimorfizmom v genu CASP-9 in trajanjem bolezni, vendar samo v skupini bolnikov, ki so bili dobri odzivniki na adalimumab. Bolniki s T/T genotipom v skupini dobrih odzivnikov so imeli dalj časa diagnosticirano bolezen kot bolniki z genotipom C/C ali C/T (p = 0,002). Boljši odziv na adalimumab je bil tudi pri mlajših bolnikih ne glede na genotip. Naša študija je pokazala, da bi analiza polimorfizma v genu CASP-9 pomagala pri napovedi odziva Crohnovih bolnikov na adalimumab, analiza genov CASP-9 in FCGR3A pa pri napovedi odziva na standardna zdravila za Crohnovo bolezen.Inflammatory bowel disease (IBD) is chronic disease with two main subtypes, Crohn disease (CD) and ulcerative colitis (UC) where several genes and environmental factors play role. Patients with Crohn disease have different response to treatment so the therapy is given in different stages. First we try 5-aminosalicylates, later corticosteroids and at the end immunosuppressors. In Crohn disease patients where standard therapy is non-responsive to treatment, the new biological therapy with monoclonal antibodies against TNF-α (adalimumab (Humira) and infliximab (Remicade)) is being used. The aim of our work was to find pharmacogenomic biomarkers which could serve as predictive factors for response to adalimumab treatment in Crohn disease patients. In our work we analyzed polymorphisms in selected apoptosis genes TIMP-1 (SNP +372C/T or rs4898), CASP-9 (SNP 93C/T or rs4645983), FCGR3A (SNP 158V/V or rs396991 and FAS (SNP -670C/T or rs1800682), which showed correlation with response to treatment with infliximab. The aim of our work was to investigate if these polymorphisms in selected apoptotic genes are associated with development of disease in Slovenian Crohn disease patients which do not respond to standard therapy or standard therapy is terminated because of severe side effects. In addition we wanted to found correlation between polymorphisms in selected genes and clinical characteristics of patients. Blood samples for DNA isolation and other analysis of biochemical and immunological parameters were taken before treatment and in 4., 12., 20 and 30. weeks after first treatment with anti TNF-α. We have genotyped single nucleotide polymorphisms (SNPs) using RFLP method followed by gel electrophoresis and KASPar technique. We found correlation between SNP in gene CASP-9 and response to treatment with adalimumab. Patients with C/C genotype had worse response to adalimumab after fourth weeks (p = 0,05) and 20. weeks (p = 0,031) of treatment compared to patients with C/T and T/T genotype. Results of our study are comparable with previous study where patients were treated with infliximab and patients with C/C genotype in CASP-9 gene had worse response to therapy. In other three genes the correlation was not statistically significant. Further we found correlation between polymorphisms in genes CASP-9 (SNP rs4645983) and FCGR3A (SNP rs396991) in Slovenian patients with CD involved in study. Frequency of allele C in SNP rs4645983 on gene CASP-9 is significantly lower in patients with CD (0,68) compared to control group (0,86, p = 9,9*10-5) as well as the frequency of C/C genotype is lower in patients with CD (60,22%) compared to control group (78,41%, p = 0,01). When comparing allele frequency of SNP rs396991 on gene FCGR3A we found statistically lower frequency of T allele in patients with CD (0,54) compared to healthy individuals (0,67, p = 0,01) as well as statistically lower frequency of T/T genotype in CD patients (33,3%) compared to control group (45,06%, p = 0,01). We also found correlation between SNP in CASP-9 and disease duration but only in a subgroup of CD patients showing good response to adalimumab. A subgroup of CD patients with good response to adalimumab and T/T genotype had longer disease duration compared to carriers of C/T or CC genotype. Younger patients had better response to adalimumab regardless the genotype they carry. Our study suggested analysis of polymorphism in CASP-9 might help to predict response to adalimumab and analysis of SNPs in CASP-9 and FCGR3A might help to predict response to standard therapy in Crohn disease patients