vicki powers bristol royal infirmary cyp2b6 g516t genotyping in patients with hiv: a...
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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
NH
O
O
Cl
CF3
efavirenz
Efavirenz (EFV) metabolism
Phase I CYP2B6
Phase II Glucuronidation
NNRTI
600/800 mg od
Therapeutic range 1-4 g/mL
Excreted
NH
O
O
Cl
CF3
NH
O
O
Cl
CF3
O-glucuronide
NH
O
O
Cl
CF3
OH
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
2 65 87 93 4
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
Males Females
Number 149 59
Age (years) 22-82 (42) 23-67 (39)
Weight (kg) 53.5-115.5 (73.8) 43.2 – 108.2 (71.9)
CD4 count (x109 cells/L)
0.045-1.416 (0.416)
0.008-1.410 (0.436)
Viral load (copies/mL)
<40–193,528 (<40)
62-741,218 (<40)
HCV coinfection 6 (3 unknown) 3 (5 unknown)
ALT (U/L) 14-145 (27) 9-456 (20)
Patient Ethnicity
Males Females
Caucasian 128 15
Black-African 18 36
Black-Caribbean 2 5
Black-South American
1 1
Asian 0 2
HAART Regimen
Males Females
Including EFV 47 (46%) 27 (32%)
Excluding EFV 85 (49%) 29 (57%)
None 17 (5%) 3 (11%)
PCR Methodology
Jacob RM Clin Chem 2004; 50:8, 1372-1377
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 (dH2O)
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12
13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24
25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 35 35 36 36
37 37 38 38 39 39 40 40 41 41 42 42 43 43 44 44 45 45 46 46 47 47 48 48
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 = 0.19 P = 0.06 P = 0.10
Questionnaire score vs HAART regimen (2)
ON EFV OTHER NONE
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)
GG GT TT
Stopped EFV 12 14 5
Remained on EFV 35 31 8
No significant difference (P = 0.63) found
Conclusions & Further Work
CYP2B6 G516T genotyping method was developed
- 208 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