Prevalence of five pharmacologically most important CYP2C9 and CYP2C19 allelic variants in the population from the Republic of Srpska in Bosnia and Herzegovina

Genetic analysis was done at the Laboratory for Molecular Biology and Genetics of the University of Banja Luka Faculty of Medicine Center for Biomedical Research. Genomic DNA was extracted from 3–6 mL of peripheral blood collected in Na-EDTA tubes using PureLink^® gDNA Blood Kit (Invitrogen, Carlsbad, CA, USA). Genotypes of CYP2C9 *2 (3608C>T, rs1799853), CYP2C9*3 (42164A>C, rs1057910 ), CYP2C19*2 (681G>A, rs4244285), CYP2C19*3 (17948G>A, rs4986893), and CYP2C19*17 (‑806C>T, rs12248560) were determined with real-time polymerase chain reaction (RT-PCR) using TaqMan^® drug metabolism genotyping assays (C_25625805_10, C_27104892_10, C_25986767_70, C_469857_10, and C_27861809_10, respectively) according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). PCR conditions included initial denaturation/polymerase activation at 95 °C for 10 min, followed by 50 two-step cycles: denaturation at 95 °C for 15 s and annealing and extension at 60 °C for 90 s. The results were analysed with the Applied Biosystems 7500 software v2.0.6 (Applied Biosystems).

Allele and genotype prevalences were estimated by gene counting. Genotype distributions were tested for the Hardy-Weinberg equilibrium. Chi-squared test with Yate’s correction was used to test similarities or differences in allele distribution. For all analyses we used the Social Science Statistics online calculators (https://www.socscistatistics.com/tests/)

As expected, the wild-type CYP2C9*1 dominated (78.90 %), followed by the CYP2C9*2 variant (13.66 %), while the prevalence of the CYP2C9*3 allele was 7.40 % (Table 1). All observed genotype frequencies were in the Hardy-Weinberg equilibrium. Accordingly, the most prevalent (62.03 %) were normal metabolisers (*1/*1 genotype). Poor metabolisers (*2/*3 and *3/*3 genotype) accounted for 1.86 %, and intermediate metabolisers (*1/*2, *1/*3 or *2/*2 genotype) for 36.11 % of participants.

The prevalences of CYP2C9*2 and CYP2C9*3 alleles found in our study are similar to the respective ones reported in other European countries such as Germany (14.0 %; p=0.29 and 5 %; p=0.37) (20), Russia (10.5 %; p=0.16 and 6.7 %; p=0.78) (22), Spain (16 %; p=0.36 and 10 %; p=0.25) (23), Greece (12.9 %; p=0.80 and 8.13 %; p=0.58) (24), North Macedonia (13.9 %; p=0.91 and 7.3 %; p=0.94) (17), Croatia (14.5 %; p=0.65 and 7.6 %; p=0.89) (8), Serbia (11.7 %; p=0.30 and 8.1 %; p=0.68) (25), and Slovenia (12.2 %; p=0.54 and 6.3 %; p=0.55) (26). However, one Romanian study (27) showed significantly higher prevalence of the *2 and *3 alleles (29 %, p<0.0001 and 20.2 %; p<0.0001, respectively). The general prevalences of the CYP2C9*2 and *3 alleles in the Caucasian population are about 13 % (*2) and 7 % (*3) (28) but significantly lower in the East Asians (0.01 % and 0.3 %), Africans (2 % and 1 %), and African-Americans (1 % and 0.5 %) (29, 30). A more detailed comparison is available in Table 3.

Again, the wild-type CYP2C19*1 allele was the most prevalent (63.43 %), followed by CYP2C19*2 (16.20 %) and CYP2C19*17 (20.37 %) (Table 2). The observed prevalence of the *2 and *17 allelic variants were within the European ranges (8, 18). Similar prevalences have been reported for the neighbouring countries. In the Serbian population the prevalence of the *2 allele was 16.3 % (p=0.97) and of the *17 allele 22.2 % (p=0.45). In the Croatian population it was 14.8 % (p=0.50) and 23.7 % (p=0.19), in the North Macedonian 14.4 % (p=0.48) and 20.1 % (p=0.93), and in the population of Kosovo 13.03 % (p=0.18) and 19.01 % (p=0.61), respectively (8, 17, 25, 30). However, Russian and Greek studies showed a significant difference in the *2 allele prevalence (p=0.0002 and p=0.0035, respectively) (22, 24). A more detailed comparison is given in Table 3.

Eighty-nine participants (41.20 %) with the CYP2C19*1/*1 genotype were normal metabolisers, 52 (24.07 %) with the CYP2C19*1/*2 and CYP2C19*2/*17 genotypes were intermediate metabolisers, nine (4.17 %) with the CYP2C19*2/*2 genotype were poor metabolisers, 57 (26.39 %) with the CYP2C19*1/*17 genotype rapid metabolisers, and nine (4.17 %) with the CYP2C19*17/*17 genotype were ultra-rapid metabolisers. Carriers of the CYP2C19*17 allele are at risk of bleeding, especially the ultra-rapid metabolisers (carriers of the CYP2C19*17/*17 genotype) (18). Carriers of the CYP2C19*2/*17 genotype are generally considered intermediate metabolisers, but their metabolic phenotype is difficult to predict, since some data suggest that CYP2C19*17 may not compensate for the CYP2C19*2 allele (8).

As for the CYP2C19*3 allele, none was detected in this study, which corresponds to other Caucasian populations such as Croatian, Danish, Canadian, German, Polish, and Australian (4, 31, 32, 33, 34, 35).