vicki powers bristol royal infirmary cyp2b6 g516t genotyping in patients with hiv: a...

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