Impact des polymorphismes génétiques du cytochrome P450 2B6 sur les concentrations plasmatiques de l’EFV et de la NVP chez les patients infectés par le virus VIH-1 au Rwanda. (Doctoral thesis)

Objectives: Over 34 million individuals are living worldwide with the human immunodeficiency virus (HIV) and the majority living in Sub-Saharan Africa (22.5 million). In Rwanda, more than 96000 patients were receiving antiretroviral drugs by 2013. The Rwandese first-line treatment is a non-nucleoside reverse transcriptase inhibitor based regimen containing either efavirenz (EFV) or nevirapine (NVP). High interindividual variations in EFV and NVP plasma concentrations have been reported which are partially caused by the high genetic variability of the cytochrome P450 2B6 (CYP2B6). The aims of this thesis project were (i) to identify new single nucleotide polymorphisms (SNP) within the CYP2B6 gene, (ii) to characterize in vitro and in silico their effects on the enzyme activity,(iii) to determine the frequency of new and known SNP in HIV-infected patients from Rwanda and (iv) to correlate the SNP/haplotypes with plasma concentrations of EFV, NVP and their main hydroxy metabolites.
Methods: The nine CYP2B6 exons of 39 individuals from Rwanda were sequenced. Eight new nonsynonymous SNP were recombinantly expressed in COS-1 cells and the new CYP2B6 variants were functionally characterized in vitro using the substrates bupropion and EFV. The results were compared with the functional prediction potency of eight algorithms. Docking and long-term molecular dynamic (MD) simulations were performed to describe structural changes and interaction modifications between the substrates and the CYP2B6 binding pocket. 19 SNP were genotyped with a MALDI-TOF genotyping method and 11 SNP were discriminated with real time PCR in 806 individuals from Rwanda. Steady-state EFV, NVP and their main hydroxy metabolites 7-OH-, 8-OH-, 8,14-OH EFV and 2-OH, 3-OH NVP were quantified using LC/MS-MS in 431 Rwandese patients under EFV or NVP therapy. Uni- and multivariate statistical analyses were performed to assess the genotype significance in EFV and NVP drug distribution.
Results: Three new [c.548T>G (p.V183G), c.637T>C (p.F213L), c.758G>A (p.R253H)] and five uncharacterized non-synonymous SNP [c.329G>T (p.G110V), c.341T>C (p.I114T), c.444G>T (p.E148D), c.835G>C (p.A279P), c.1459C>A (p.R487S)] were identified and five novel alleles termed CYP2B6*33 – CYP2B6*37 were assigned. In vitro analysis revealed that the variants 148D, 253H, 279P and 487S were functional whereas the variant 213L showed a reduced enzyme activity and the variants 110V, 114T and 183G were complete loss-of-function variants. 60% to 80% of the in silico functional predictions of each variant were correct. The MD simulations showed that only the variants 110V, 114T, 183G and 213L had structural modifications in alpha-helices and beta -strands surrounding the catalytic site of the CYP2B6 enzyme. The frequency of the eight polymorphisms in the Rwandese population was <0.9% classifying them as rare variants. 71% of 219 EFV-treated patients and 42% of 212 NVP-treated patients had supra- and subtherapeutic plasma concentrations, respectively. A new allele termed CYP2B6Rwa7 (SNP c.341T>C, c.444G>T, c.835G>C) was significantly associated to low EFV and NVP concentrations. The “CYP2B6-TGT” haplotype (c.516T, c.785G, g.21563T) was strongly related to high EFV and NVP and low 8-OH EFV and 3-OH NVP plasma concentrations. The *6/*18 genotype, the homozygous g.21563TT SNP and the homozygous TGT haplotype were found as the most relevant independent factors to account for high EFV concentrations.
Conclusions: We have described three novel SNP with two altering the CYP2B6 enzyme function in vitro and assigned five new CYP2B6 alleles in a Rwandese population. Our research work allowed us to identify a new CYP2B6 allele which was correlated to low EFV and NVP concentrations and to confirm the existence of genetic markers predicting supra-therapeutic EFV plasma concentrations. Our results suggest an individualized genotyping strategy to sustain the efficiency and the durability of the antiretroviral therapy in Sub-Saharan Africa.