Karakteristike distribucije funkcionalnih varijanti gena iz superfamilije citohroma P450 u stanovništvu Srbije

Abstract

Citohrom P450 genska superfamilija se u ljudskom genomu sastoji od 57 gena, koji se na osnovu aminokiselinske sličnosti razvrstavaju u 44 genske podfamilije i 18 familija. Geni koji pripadaju ovoj familiji se prevode u enzime sa raznovrsnim funkcijama, koji se nazivaju CYP enzimi i eksprimiraju se u različitim tkivima u ljudskom organizmu. Među najznačajnijim funkcijama CYP enzima su sposobnost katalize oksidacije, redukcije i hidroksilacije, zbog čega se ovi enzimi nazivaju i monooksigenazama i poznato je da učestvuju u fazi I metabolizma ksenobiotika. U širokom spektru ksenobiotika koji se metabolišu CYP enzimima se nalazi i veliki broj lekova koji su u kliničkoj upotrebi, što omogućava proučavanje uticaja genetičke varijabilnosti na farmakodinamiju i farmakokinetiku lekova. Uticaj genetičkih promena u CYP genima se prvenstvno ogleda u izmeni aminokiselinske sekvence, a posledično i u smanjenoj funkciji CYP enzima. Pored toga, izmene u DNK sekvenci koja reguliše ekspresiju CYP gena, mogu dovesti do umanjena ili povećanja kapaciteta CYP enzima da metabolišu lek. Izmene u CYP genima, koje najćešće dovode do promena u funkciji CYP enzima, se nazivaju funkcionalne varijante i mogu biti tačkaste izmene ili inserciono-delecione varijante u DNK sekvenci. Na osnovu genotipizacije funkcionalnih varijanti CYP gena, moguće je odrediti i funkcionalnost enzima, kao i metabolički kapacitet čovekovog organizma prema leku za koji je poznato da zavisi od funkcionalnosti tog enzima. Cilj je precizniji odabir vrste i doze leka, kao i izbegavanje neželjene reakcije na lek. Ovakav pristup se naziva farmakogenetikom i predstavlja osnovu na kojoj se razvija personalizovana medicina i molekularna autopsija. Od svih CYP genskih familija koje su bitne za metabolizam lekova, familije CYP1, CYP2 i CYP3 imaju najvećeg uticaja i najbolje su istražene, a unutar njih postoje genetičke varijante za koje je dokumentovano da dovode do izmena u metabolizmu lekova. Međutim, ciljana primena molekularno bioloških metoda za genotipizaciju zahteva prethodno znanje o informativnosti CYP lokusa, koja može u velikoj meri zavisiti od učestalosti genetičkih varijanti u nekoj populaciji, što nameće potrebu da se istraživanje CYP varijanti sprovede na populaciono genetičkom nivou. Kako je u ovom trenutku informacija o karakteristikama raspodele učestalosti CYP funkcionalnih varijanti u stanovništu Srbije oskudna i nesistematična, ova studija je imala za cilj da u uzorku koji će verodostojno predstaviti opštu populaciju Srbije, odredi učestalosti alela i genotipova 11 varijanti u CYP genima: CYP1A1 ( rs4646903, rs1048943), CYP2C9 (rs1057910, rs1799853), CYP2C19 (rs12248560, rs4244285), CYP2D6 (rs3892097, rs1065852, rs28371706, rs28371725), CYP3A4 (rs2740574). Pored određivanja učestalosti funkcionalnih varijanti, jedan od ciljeva je bio i da se analizira gentička struktura stanovništva pomoću formiranja uzorka od osoba koje pripadaju potencijalno najdiferenciranijoj subpopulaciji i upotrebom selektivno neutralnih mikrosatelitskih lokusa, kao i većeg broja algoritama za detekciju genetičke struktuiranosti. Radi povećanja reprezentativnosti uzorka opšte populacije, odabran je metod stratifikovanog uzorkovanja DNK izolata nesrodnih osoba prema mestu prebivališta iz pet regiona Srbije: Severna Srbija (Vojvodina), okrug Beograda, Zapadna Srbija, Centralna Srbija, Južna i Istočna Srbija. Ukupan uzorak je iznosio 550 izolata, dok je u svojstu uzorka iz potencijalno najdiferenciranije subpopulacije, odabrano 60 DNK izolata od osoba romske nacionalne pripadnosti. Svi uzorci su genotipizirani primenom lančane reakcije polimeraze u realnom vremenu (rs1057910, rs1799853,rs12248560, rs4244285, rs3892097, rs1065852, rs28371706, rs28371725, rs2740574) i analizom polimorfizama dužine restrikcionih fragmenata (rs4646903, rs1048943). Na podacima uspešno genotipiziranih ispitanika sprovedena je bioinformatička analiza upotrebom algoritma koji se zasnivaju na Bejzovom pristupu statističkog zaključivanja, redukciji dimenzionalnosti podataka, hijerarhijskom klasterovanju, kao i primeni teorije mreža u analizi podataka...

The cytochrome P450 gene superfamily in the human genome consists of 57 genes, which are classified into 44 gene subfamilies and 18 families based on amino acid similarity. Genes belonging to this family are translated into enzymes with various functions, called CYP enzymes and are expressed in various tissues in the human body. Among the most important functions of CYP enzymes is the ability to catalyze oxidation, reduction and hydroxylation reactions, which is why these enzymes are also called monooxygenases and are known to participate in phase I metabolism of xenobiotics. The wide range of xenobiotics that are metabolized by CYP enzymes also includes a large number of drugs that are in clinical use, which enables the study of the influence of genetic variability on the pharmacodynamics and pharmacokinetics of drugs. The influence of genetic changes in CYP genes is primarily reflected in the change of the amino acid sequence, and consequently in the reduced function of CYP enzymes. Additionally, changes in the DNA sequence, that regulate CYP gene expression, may lead to a decrease or increase in the capacity of CYP enzymes to metabolize a drug. Changes in CYP genes, which most often lead to changes in the function of the CYP enzymes, are called functional variants and can be point changes or insertion-deletion variants in the DNA sequence. Based on the genotyping of functional variants of a CYP gene, it is possible to determine the functionality of the enzyme, as well as the metabolic capacity of the human body for a drug known to depend on the functionality of the enzyme. The goal is to accurately choose the type and dose of the drug, as well as to avoid an adverse reaction to it. This approach is called pharmacogenetics and is the basis for development of personalized medicine and molecular autopsy. Of all the CYP gene families important for drug metabolism, the CYP1, CYP2, and CYP3 families have the greatest impact, are most researched and encompass genetic variants that have been documented to lead to changes in drug metabolism. However, the targeted application of molecular biology methods for genotyping requires prior knowledge of the informativeness of the CYP locus, which may largely depend on the frequency of genetic variants in a population, necessitating research on CYP variants at the population genetic level. As information on the characteristics of the frequency distribution of CYP functional variants in the population of Serbia is scarce and unsystematic, this study aimed to determine the frequencies of alleles and genotypes of 11 variants in CYP genes in a sample that will credibly represent the general population of Serbia: CYP1A1 (rs4646903, rs1048943), CYP2C9 (rs1057910, rs1799853), CYP2C19 (rs12248560, rs4244285), CYP2D6 (rs3892097, rs1065852, rs28371706, rs28371725), CYP3A4 (rs2740574). In addition to determining the frequency of functional variants, one of the goals was to analyze the genetic structure of the population by forming a sample of people belonging to the potentially most differentiated subpopulation and using selectively neutral microsatellite loci, as well as a number of algorithms for detecting genetic structure. In order to increase the representativeness of the general population sample, the chosen method was the one of stratified sampling of DNA isolates of unrelated persons according to the place of residence from five regions of Serbia: Northern Serbia (Vojvodina), Belgrade district, Western Serbia, Central Serbia, Southern and Eastern Serbia. The total sample was 550 isolates, while 60 DNA isolates of persons of Roma ethnicity were selected as a sample from the potentially most differentiated subpopulation. All samples were genotyped using real-time polymerase chain reaction (rs1057910, rs1799853, rs12248560, rs4244285, rs3892097, rs1065852, rs28371706, rs28371725, rs2740574) and analysis of restriction fragment length polymorphism (rs4646903, rs1048943). Bioinformatics analysis was performed on the data of successfully genotyped subjects using algorithms based on Bayes' approach to statistical inference, dimensionality reduction, hierarchical clustering, as well as network theory...