Vicki Powers Bristol Royal Infirmary CYP2B6 G516T genotyping in patients with HIV: A pharmacogenetics study of the antiretroviral, efavirenz

HIV replication cycle Weiss RA. EMBO reports 4, Supp1, S10–S14 (2003) X efavirenz

Efavirenz (EFV) metabolism Phase I CYP2B6 Phase II Glucuronidation NNRTI 600/800 mg od Therapeutic range 1-4  g/mL Excreted

Literature Background Haas DW Aids 2004 Tsuchiya K Biochem Biophys Res Commun 2004 Rodriguez-Novoa S Clin Infect Dis 2005 Ribuado HJ Clin Infect Dis 2006  Inter-patient variability in response to EFV  Differences seen between ethnic groups  CNS side effects and rash development reported in ~1/2 of patients  EFV discontinued in ~10% of patients due to side effects  Many studies looked at establishing a genetic cause CYP2B6 G516T  G516T homozygotes – elevated (> 4  g/mL) EFV levels  Wildtype homozygotes – subtherapeutic (< 1  g/mL) EFV levels

1 1 1 CYP2B6 G516T CYP2B6CYP2B7 Gln172His  Polymorphism frequency? ~ 3% ‘European Americans’ ~ 20% ‘African Americans’ 1

Project aims 1. To develop a PCR based genotyping method to test for the CYP2B6 G516T polymorphism 2. To establish the frequency of the CYP2B6 G516T polymorphism in a UK cohort of HIV patients 3. To correlate genotype with phenotype - side effect questionnaire 4. To look at EFV discontinuation and compare with CYP2B6 G516T genotypes

Patient recruitment  Ethical approval obtained (September 06)  Patients personally recruited at clinic  3 afternoon clinics per week (~ 50 patients seen)  30 per week  Patients were asked: 1. Consent for spare blood sample (CD4 count) to be used 2. To complete a questionnaire on side effects experienced (ACTG A5097s trial: Clifford et al. Ann Intern Med, 2001)

Questionnaire  34 questions  Questions relate to side effects reported with EFV use  Patients asked to grade how much of each experience they get: Not at all (0) A little (1) Moderately (2) Quite a bit (3) Extremely (4) Symptom score (out of 136)

Sample collection  232 patients recruited (208 analysed)  Genomic DNA was extracted from CD4 count samples using versaGene Genomic DNA purification kits (Gentra) within 5 days of blood being taken  Samples stored at –40 ºC until analysis

Patient Cohort MalesFemales Number14959 Age (years)22-82 (42)23-67 (39) Weight (kg) (73.8)43.2 – (71.9) CD4 count (x10 9 cells/L) (0.416) (0.436) Viral load (copies/mL)<40–193,528 (<40)62-741,218 (<40) HCV coinfection6 (3 unknown)3 (5 unknown) ALT (U/L) (27)9-456 (20)

Patient Ethnicity MalesFemales Caucasian12815 Black-African1836 Black-Caribbean25 Black-South American11 Asian02

HAART Regimen MalesFemales Including EFV47 (46%)27 (32%) Excluding EFV85 (49%)29 (57%) None17 (5%)3 (11%)

PCR Methodology Jacob RM Clin Chem 2004; 50:8, Step 1: Routine PCR control

Step 2: Allele specific PCR (asPCR) Rxn 1 Rxn 2 PCR Methodology GG GT TT CON asPCR prod G T

DNA Sequencing GG GT TT

Study samples  44 samples per batch, 3 +ve controls (GG, GT, TT), 1 –ve control (dH 2 O) GG GT TT -ve  3 batches (136 samples) OK…

CYP2B6 G516T Frequency TT 6% TT 16% P<0.05 (  2 )

Questionnaire Analysis (1)

Questionnaire Analysis (2) P = 0.67  Questionnaire score vs HAART regimen (1)  No significant difference in QS between HAART regimen groups

Questionnaire Analysis (3) P = 0.19P = 0.06P = 0.10  Questionnaire score vs HAART regimen (2) ON EFVOTHERNONE  No significant difference in QS between genotype groups when analysed according to HAART regimen

Discontinuation Study  Distribution of genotypes between individuals which had stopped taking EFV as part of their HAART regimen (n = 31) were compared with those who had remained on EFV (n = 74) GGGTTT Stopped EFV12145 Remained on EFV35318  No significant difference (P = 0.63) found

Conclusions & Further Work  CYP2B6 G516T genotyping method was developed study participants were genotyped - However…  Genotype-phenotype associations did not show this test would be useful in pre-treatment screening - Further work is required to investigate this

Acknowledgements Dr Mark Gompels John Ward Ann Bowron Dr Paul Thomas Immunology dept, Southmead Clinic staff and patients