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CILJ ISTRAŽIVANJA Cilj je ovoga specijalističkog rada prikupiti i kritički procijeniti relevantne podatke koji se odnose na utjecaj genskih varijanti na ishode terapije 5-fluorouracilom i irinotekanom, prikazati upute i smjernice izdane od međunarodnih tijela, te prisutne dileme u liječenju. MATERIJALI I METODE Za potrebe istraživanja i pisanja rada od dostupnih baza podataka pretraživane su baze podataka Medline i PubMed prema ključnim riječima: 5-fluorouracil, dihidropirimidin-dehidrogenaza, farmakogenetika, genski polimorfizmi i irinotekan. Odabir znanstvenih članaka publiciranih u posljednjem desetljeću obuhvaća važnije članke iz područja farmakogenetike, farmakogenomike i farmakoterapije, starije i novije objave, originalne članke kao i preglede koji zadovoljavaju tematiku i ciljeve istraživanja postavljene u radu. U radu su također prikazani i podatci o implementaciji testiranja DPYD i UGT1A1 u Hrvatskoj dobiveni u okviru doktorske disertacije dr.sc. Ivana Bilića 'Uloga polimorfizama gena dihidropirimidin-dehidrogenaze i UDP-glukuronil-transferaze u toksičnosti kemoterapije fluoropirimidinima i irinotekanom'. U tu svrhu prikazan je pregled rutinskih analiza pacijenata liječenih 5-fluorouracilom i irinotekanom tijekom tri godine (2013.-2016.) u Kliničkom zavodu za laboratorijsku dijagnostiku u Kliničkom bolničkom centru Zagreb. RASPRAVA Bolesnici s toksičnim nuspojavama na fluoropirimidine i irinotekan češće su nositelji polimorfizama gena za dihidropirimidin-dehidrogenazu, između ostalih to su DPYD*2A (c.1905+1G>A ili IVS 14+1 G>A), DPYD 496A>G (c.496A>G), te polimorfizma UGT1A1 (UGT1A1*28), u odnosu na pacijente koji nisu iskusili nuspojave. Na temelju farmakogenetičkih analiza može se u značajnoj mjeri predvidjeti razvoj nuspojava, individualizirati, tj. prilagoditi doza lijeka i time minimalizirati rizik od nastanka štetnih učinaka. ZAKLJUČAK Iako postoje jasni dokazi o vrijednosti farmakogenetičkih testiranja, njihova implementacija u svakodnevnu kliničku praksu zaostaje za tim spoznajama. Poznavanje genskih polimorfizama ne može apsolutno predvidjeti sve neočekivane reakcije pacijenta ali može značajno doprinijeti individualizaciji terapije te minimalizaciji rizika. OBJECTIVES The purpose of this specialist thesis is to collect and critically evaluate relevant data related to the influence of 5-fluorouracil and irinotecan gene variants on the outcome of therapy, to present current guidelines issued by relevant international bodies, and dilemmas in the treatment. MATERIALS AND METHODS For research and writing purposes, Medline and PubMed databases were searched for by following keywords: 5-fluorouracil, dihydropyrimidine-dehydrogenase, pharmacogenetics, gene polymorphisms and irinotecan. The selection of scientific articles published in the last decade includes the most important articles in the field of of pharmacogenetics, pharmacogenomics and pharmacotherapy, older and newer releases, original articles and reviews that meet the topic and goals of the research set in this thesis. This paper presents the latest data on the implementation of DPYD and UGT1A1 testing in Croatia obtained within the doctoral dissertation of Ivan Bilić 'The role of dihydropyrimidinedehydrogenase and UDP-glucuronyl-transferase polymorphisms in fluoropyrimidine and irinotecan toxicity'. For this purpose, the routine analyses of patients treated with 5-fluorouracil and irinotecan during the three years period (2013.-2016.), at the Clinical Institute for Laboratory Diagnosis at Zagreb Clinical Hospital Center have been presented. DISCUSSION Patients with toxic side effects of fluoropyrimidines and irinotecan are more frequent carriers of dihydropyrimidine-dehydrogenase genomic polymorphisms, among others DPYD*2A (1905 + 1G> A or IVS 14+1G> A), DPYD 496A> G (c.496A> G) and UGT1A1 polymorphism (UGT1A1*28), compared to patients who do not experience side effects. Based on pharmacogenetic analysis, it is possible to significantly anticipate the development of side effects, to individualize, i.e. to adjust the dose of the drug and minimize the risk of side effects. CONCLUSION Although there is a clear evidence of pharmacogenetic testing values, the implementation in everyday clinical practice is lagging behind these findings. Knowledge of gene polymorphisms cannot predict all unexpected responses of the patient, but can significantly contribute to the individualization of the therapy and minimizing the risk.

CYP2C9 jedan je od najvažnijih i najobilnije eksprimiranih enzima sustava citokrom P450 (CYP) u ljudskim jetrima. Odgovoran je za biotransformaciju oko 15 % klinički važnih lijekova, uključujući kumarinske antikoagulante (varfarin), antikonvulzive (fenitoin, valproična kiselina), oralne antidijabetike (gliklazid, tolbutamid), blokatore angiotenzinskih-II receptora (kandesartan, losartan), nesteroidne protuupalne lijekove (NSAID) te fluvastatin i siponimod. Gen CYP2C9 visoko je polimorfan te je, do danas, identificirano preko 60 različitih alela, među kojima najmanje njih 20 dokazano mijenja aktivnost genskog produkta, što doprinosi velikoj interindividualnoj varijabilnosti terapijskog odgovora. S obzirom na učinak polimorfizama na smanjenu aktivnost enzima te njihovu relativno veliku učestalost u bjelačkoj populaciji, najvažniji su haplotipovi CYP2C9*2 i CYP2C9*3. Navedeni aleli rezultiraju smanjenim metabolizmom većine supstrata CYP2C9 i većom izloženosti lijeku te posljedično povećanim rizikom razvoja neželjenih reakcija, osobito kod primjene lijekova s uskim terapijskim rasponom. U svrhu otkrivanja pojedinaca s povećanim rizikom razvoja neželjenih reakcija i prije početka liječenja, provodi se genotipizacija CYP2C9 na temelju koje se zaključuje o metaboličkom fenotipu. Tri su moguća fenotipa CYP2C9: spori (PM), intermedijarni (IM) i normalni (NM) metabolizatori. U bjelačkoj populaciji spori metabolizatori čine 3-5 %. Ovakav personalizirani pristup omogućuje pravovremenu primjenu odgovarajuće terapije i bolji ishod za bolesnika. U ovome su radu prikazana dosadašnja saznanja o utjecaju genskih varijacija CYP2C9 na terapijski učinak i sigurnost lijekova-supstrata CYP2C9 te je opisan praktični pristup validaciji metode za genotipizaciju CYP2C9 u kliničkoj praksi. CYP2C9 is one of the most important and abundantly expressed enzymes of the cytochrome P450 system in the human liver. It is responsible for the metabolism of approximately 15 % of clinically important drugs, including coumarin anticoagulants (warfarin), anticonvulsants (phenytoin, valproic acid), oral antidiabetic agents (gliclazide, tolbutamide), angiotensin II receptor antagonists (candesartan, losartan), nonsteroidal anti-inflammatory drugs (NSAIDs), fluvastatin and siponimod. CYP2C9 gene is highly polymorphic and, to this day, over 60 different alleles have been identified, among which at least 20 have been proven to alter its gene product activity, which contributes to significant inter-individual variability of therapeutic response. Due to the decreasing effect on enzyme activity and the relatively high prevalence in Caucasians, CYP2C9*2 and CYP2C9*3 are the most important haplotypes. These alleles result in reduced metabolism for most CYP2C9 substrates, increased drug exposure, and consequently increased risk of adverse drug reactions, especially when administered drugs have a narrow therapeutic index. To identify individuals with increased risk of adverse drug reactions (ADRs) even before the treatment initiation, preemptive CYP2C9 genotyping is performed, and metabolic phenotype is inferred based on these results. There are three possible CYP2C9 phenotypes: poor metabolizer (PM), intermediate metabolizer (IM), and normal metabolizer (NM). In the Caucasian population, poor metabolizers make 3-5 %. This personalized approach enables in time selection of appropriate therapy and a better outcome for a patient. In this paper, current knowledge on the influence of CYP2C9 gene variations on the efficacy and safety of drugs metabolized by the CYP2C9 enzyme and a practical approach to validation of methods for CYP2C9 genotyping in clinical practice is presented.

Metabolički sindrom (MetS) je poremećaj za čiji razvoj je odgovoran cijeli niz čimbenika kao što su dislipidemija, abdominalna pretilost, hipertenzija i hiperglikemija. U čimbenike rizika ubrajaju se i genetički čimbenici. Kako bi se utvrdilo da li promjene u strukturi gena za apolipoprotein A5 (ApoA5) i protein-nosač masnih kiselina 2 (FABP2) koji sudjeluju u izvanstaničnom/unutarstaničnom transportu lipida utječu na nastanak MetS u starijoj populaciji, provedeno je istraživanje na 345 ispitanika muškog i ženskog spola starosti od 70 do 93 godine. Za svakog su ispitanika utvrđene antropometrijske vrijednosti, a biokemijskim su analizama određeni parametri lipidnog statusa u serumu, kao i koncentracija glukoze, ukupni proteini i C-reaktivni protein. Genotipizacija odabranih gena provedena je metodama temeljenim na lančanoj reakciji polimerazom. Plinskom kromatografijom određen je sastav i koncentracije masnih kiselina u ispitanika s i bez MetS. Statističkom je analizom utvrđeno da genske varijante gena za ApoA5 i gena za FABP2 nisu povezane s povećanim rizikom za razvoj metaboličkog sindroma. Međutim, polimorfizmi gena za ApoA5 i gena za FABP2 mogu biti povezani s koncentracijama lipida u serumu starije populacije s metaboličkim sindromom. Zaključak je da bi se stoga ApoA5 i FABP2 mogli razmotriti kao čimbenici patobiokemije multifaktorijalnih poremećaja poput MetS. Metabolic syndrome (MetS) is a multifactorial disorder. Among the risk factors for developing MetS are dyslipidemia, abdominal obesity, hypertension and hyperglycemia. The other risk factors include also the genetic factors. To determine whether changes in the structure of the gene for apolipoprotein A5 (ApoA5) and the fatty acid-binding protein 2 (FABP2), that participate in the extracellular / intracellular transport of lipids influence the occurrence of MetS in older population, an investigation was conducted on 345 subjects, males and females, aged 70-93 years. For each subject were determined the anthropometric values. The concentrations of glucose, total protein and C-reactive protein were determined in serum by biochemical methods. Genotyping of selected genes was carried out by methods based on the polymerase chain reaction. The composition and concentration of fatty acids in subjects with and without MetS were determined by gas chromatography. Statistical analysis showed that genetic variants of ApoA5 gene and FABP2 gene were not associated with an increased risk for developing metabolic syndrome. However, ApoA5 and FABP2 gene polymorphisms may be associated with serum lipid levels in an elderly population with MetS. The conclusion is that the ApoA5 and FABP2 could be considered as factors for pathobiochemistry of multifactorial disorders such as MetS.

Cerebrovaskularni poremećaji (CVP) su značajan medicinski problem u djece i odraslih. Cilj istraživanja bio je ispitati razlike u učestalostima polimorfizama gena za HPA (-1, -2, -3, -5), PSEL (S290N, N562D, V599L, T715P), mutacija FV G1691A (FVL) i FII G20210A u bolesnika s CVP-om u odnosu na kontrolnu skupinu, te utvrditi njihovu povezanost s bolešću. Ukupno je ispitano 300 bolesnika s CVP-om (ishemijski moždani udar-iCVI, TIA) (djece-100, odraslih-200) i 200 ispitanika kontrolne skupine (djece-100, odraslih-100), uz podatke za odrasle o standardnim čimbenicima rizika. Polimorfizmi HPA genotipizirani su PCR-SSP-om i PCR-om u stvarnom vremenu, PSEL-a PCR-SSP-om, a FVL i FII G20210A PCR-RFLP-om. U Hrvata alelne učestalosti HPA odgovaraju podacima za Europljane. Multivarijantnom regresijskom analizom u odraslih potvrđena je povezanost anamneze, hipertenzije, šećerne bolesti i alkoholizma s CVP-om, ali ne s alelom HPA-2b. U djece značajno je učestaliji genotip HPA-5a/b i alel HPA-5b u oboljelih od TIA-e u odnosu na iCVI, a u djece s genotipom GA ili s alelom A FVL, 5 puta je veća vjerojatnost oboljenja od CVP-a. Značajno učestaliji genotip GA i alel A FVL dobiven je u djece u odnosu na odrasle s CVP-om, te iCVI-om. Pomoć u predviđanju CVP-a u odraslih imaju anamneza, hipertenzija, šećerna bolest i alkoholizam, a u djece FVL. Cerebrovascular disorders (CVD) are a significant medical problem in children and adults. The aim of this research was to examine differences in the frequency of gene polymorphisms for HPA (-1, -2, -3, -5), PSEL (S290N, N562D, V599L, T715P), FV G1691A (FVL) and FII G20210A mutations in patients with CVD as compared to the control group, and to determine their correlation with the disease. The research included 300 patients with CVD (ischemic stroke-iCVI, TIA) (children-100, adults-200), 200 control subjects (children-100, adult-100), as well as data for standard risk factors for adults. HPA polymorphisms were genotyped using PCR-SSP and real-time PCR, PSEL by PCR-SSP, and the FVL and FII G20210A by PCR-RFLP. The allelic frequencies of HPA in Croats correspond to the data for Europeans. Multivariate regression analysis in adults confirmed the correlation between family history, hypertension, diabetes and alcoholism with CVD, but not with the HPA-2b allele. In children, HPA-5a/b genotype and HPA-5b allele is significantly more frequent in patients with TIA as compared to iCVI, and in children with GA genotype or with A allele FVL, there is a 5 times higher likelihood of CVD disease. Significantly more frequent GA genotype and A allele FVL was obtained in children than in adults with CVD and iCVI. Help in predicting CVD in adults is provided by family history, hypertension, diabetes and alcoholism, and in children by FVL.