| Abstract | Sustav citokroma P450 uključen je u biotransformaciju >75% lijekova na tržištu pri čemu se oko
20% njih metabolizira s pomoću enzima CYP2D6. Gen CYP2D6 izrazito je varijabilan, do sad je
detektirano >170 varijanti, uključujući polimorfizme jednog nukleotida (SNP) i strukturne varijante
(SV). Od strukturnih varijanti javljaju se delecije, duplikacije i multiplikacije te hibridni geni
CYP2D6::CYP2D7 i CYP2D7::CYP2D6. Različite varijante gena CYP2D6 kodiraju enzime
CYP2D6 različite aktivnosti pa se pacijenti u ovisnosti o genotipu svrstavaju u fenotipske skupine
normalnih metabolizatora, intermedijarnih, sporih i vrlo brzih. Ispravna genotipizacija i
fenotipizacija ključne su za doziranje lijekova čiji učinci primarno ovise o metabolizmu putem
enzima CYP2D6, posebno ako se radi o lijekovima s uskim terapijskim rasponom. Pacijenti s
različitim fenotipom zahtijevaju različite doze takvih lijekova kako bi se s jedne strane izbjeglo
poddoziranje i izostanak željenog učinka, a s druge strane predoziranje i neželjeni učinci.
S obzirom na visoku varijabilnost gena, genotipizacija CYP2D6 je kompleksna. Poseban izazov
predstavlja detekcija hibridnih gena. PCR metode za umnažanje dugih odsječaka DNA (XL-PCR)
te TaqMan metode uspješno detektiraju SNP-ove, ali nemaju mogućnost detekcije hibridnih gena,
već se oni lažno detektiraju kao cjelovite varijante ili kao potpune delecije gena CYP2D6. CNV
analize (od eng. copy number variation), usmjerene na 2 ili više fragmenta gena, donose revoluciju
u prepoznavanju hibrida.
U ovom radu opisan je postupak farmakogenetske analize gena CYP2D6 koja uključuje analizu
polimorfizama jednog nukleotida i CNV analizu, s naglaskom na analizu strukturnih varijanti i
problematiku genotipizacije hibridnih gena. Prikazan je pregled zastupljenosti različitih alela,
genotipa i fenotipa u populaciji pacijenata upućenih na genotipizaciju na Odjel za farmakogenomiku
i individualizaciju terapije Kliničkog zavoda za laboratorijsku dijagnostiku Kliničkog bolničkog
centra Zagreb te su uspoređeni rezultati dobiveni s CNV analizom i bez nje kako bi se dobio uvid u
značaj CNV analize. Opisani su i problemi na koje nailazi CNV analiza. Njena kompleksnost
proizlazi iz činjenice da sami hibridni geni dolaze u velikom broju raznolikih varijanti te su nerijetko
uključeni u neidentične duplikacije.
U konačnici, može se reći da CNV analiza povećava kvalitetu farmakogenetske analize, međutim
metoda nije savršena. Stoga ostaju otvorena pitanja optimiranja CNV analize i mogućnosti uvođenja
drugih molekularnih metoda genotipizacije gena CYP2D6 u kliničku praksu. |
| Abstract (english) | Cytochrome P450 system is involved in the biotransformation of >75% of the approved drugs,
where about 20% of them are metabolized by CYP2D6 enzyme. The CYP2D6 gene is extremely
variable, so far >170 variants have been detected, including single nucleotide polymorphisms (SNPs)
and structural variants (SVs). Structural variants include deletions, duplications and multiplications
and also hybrid genes CYP2D6::CYP2D7 and CYP2D7::CYP2D6. Different variants of the CYP2D6
gene encode CYP2D6 enzymes with different activities, so depending on the genotype, patients are
classified into phenotypic groups of normal, intermediate, poor and ultrarapid metabolizers.
Correctly determined genotype and phenotype are crucial for the dosing of drugs whose effects
primarily depend on metabolism via the CYP2D6 enzyme, especially for narrow therapeutic index
drugs. Patients with different phenotypes require different doses of such drugs in order to avoid on
the one hand underdosing and absence of the desired effect, and on the other hand, overdosing which
leads to side effects and toxicity.
Due to the variability of CYP2D6 gene, genotyping is complex. The detection of hybrid genes is
particulary challenging. Extra-long Polymerase Chain Reaction (XL-PCR) and TaqMan® real-time
PCR method successfully detect SNPs, but do not have the possibility of detecting hybrid genes,
instead they are falsely detected as whole genes or as deletions. CNV analysis, that detects 2 or more
CYP2D6 gene fragments, revolutionize the discovery of hybrid genes.
This paper describes the procedure of pharmacogenomic analysis of the CYP2D6 gene, which
includes the analysis of single nucleotide polymorphisms and CNV analysis, with an emphasis on
the analysis of structural variants and the difficulties in genotyping hybrid genes. An overview of the
frequencies of different allels, genotypes and fenotypes in the population of patients who underwent
pharmacogenomic analysis in the Division for Pharmacogenomics and Therapy Individualization of
the Department for Laboratory Diagnostics University Hospital Center Zagreb is presented. Also the
results of genotyping and phenotyping obtained with and without CNV analysis are compared in
order to gain insight into the significance of CNV analysis. The weaknesses of CNV analysis are also
described. Its complexity stems from the fact that the hybrid genes themselves come in a large
number of diverse variants and are often involved in non-identical duplications.
Ultimately, it can be said that CNV analysis increases the quality of pharmacogenomic analysis
of the CYP2D6 gene, however the method is not perfect. Therefore, the questions of optimizing CNV
analysis and the possibility of introducing other molecular methods for genotyping the CYP2D6 gene
into clinical practice remain open. |
