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

Objectives: Over 34 millionindividuals are living worldwide with the human immunodeficiency virus (HIV)and the majority is living in Sub-Saharan Africa (22.5 million). In Rwanda,more than 96000 patients were receiving antiretroviral drugs by 2013. TheRwandese first-line treatment is a non-nucleoside reverse transcriptaseinhibitor based regimen containing either efavirenz (EFV) or nevirapine (NVP).High interindividual variations in EFV and NVP plasma concentrations have beenreported which are partially caused by the high genetic variability of thecytochrome 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 theenzyme activity, (iii) to determine the frequency of new and known SNP inHIV-infected patients from Rwanda and (iv) to correlate the SNP/haplotypes withplasma concentrations of EFV, NVP and their main hydroxy metabolites.

Methods: The nine CYP2B6 exonsof 39 individuals from Rwanda were sequenced. Eight new nonsynonymous SNP wererecombinantly expressed in COS-1 cells and the new CYP2B6 variants were functionallycharacterized in vitro using the substrates bupropion and EFV. Theresults were compared with the functional prediction potency of eightalgorithms. Docking and long-term molecular dynamic (MD) simulations wereperformed to describe structural changes and interaction modifications betweenthe substrates and the CYP2B6 binding pocket. 19 SNP were genotyped with aMALDI-TOF genotyping method and 11 SNP were discriminated with real time PCR in806 individuals from Rwanda. Steady-state EFV, NVP and their main hydroxymetabolites 7-OH-, 8-OH-, 8,14-OH EFV and 2-OH, 3-OH NVP were quantified usingLC/MS-MS in 431 Rwandese patients under EFV or NVP therapy. Uni- andmultivariate statistical analyses were performed to assess the genotypesignificance 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 uncharacterizednon-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 andfive novel alleles termed CYP2B6*33 – CYP2B6*37 were assigned. Invitro analysis revealed that the variants 148D, 253H, 279P and 487S were functionalwhereas the variant 213L showed a reduced enzyme activity and the variants110V, 114T and 183G were complete loss-of-function variants. 60% to 80% of the insilico functional predictions of each variant were correct. The MDsimulations showed that only the variants 110V, 114T, 183G and 213L had structuralmodifications in α-helicesand β-strands surrounding thecatalytic site of the CYP2B6 enzyme. The frequency of the eight polymorphismsin 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- andsubtherapeutic plasma concentrations, respectively. A new allele termed CYP2B6Rwa7(SNP c.341T>C, c.444G>T, c.835G>C) was significantly associated tolow EFV and NVP concentrations. The “CYP2B6- TGT” haplotype (c.516T,c.785G, g.21563T) was strongly related to high EFV and NVP andlow 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 themost relevant independent factors to account for high EFV concentrations.

Conclusions: We have described three novel SNP with two alteringthe CYP2B6 enzyme function in vitro and assigned five new CYP2B6 allelesin a Rwandese population. Our research work allowed us to identify a new CYP2B6allele which was correlated to low EFV and NVP concentrations and toconfirm the existence of genetic markers predicting supra-therapeutic EFVplasma concentrations. Our results suggest an individualized genotypingstrategy to sustain the efficiency and the durability of the antiretroviral therapyin Sub-Saharan Africa.