Vicki Powers

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CYP2B6 G516T genotyping in patients with HIV A
pharmacogenetics study of the antiretroviral,
efavirenz
Vicki Powers Bristol Royal Infirmary
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HIV replication cycle
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Efavirenz (EFV) metabolism
NNRTI 600/800 mg od Therapeutic range 1-4 ?g/mL
Phase I CYP2B6
Excreted
Phase II Glucuronidation
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Literature Background

• 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

Haas DW Aids 2004 Tsuchiya K Biochem Biophys Res
Commun 2004 Rodriguez-Novoa S Clin Infect Dis
2005 Ribuado HJ Clin Infect Dis 2006

• G516T homozygotes elevated (gt 4 ?g/mL) EFV
  levels

• Wildtype homozygotes subtherapeutic (lt 1 ?g/mL)
  EFV levels

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CYP2B6
1
1
1
1

• Polymorphism frequency? 3 European
  Americans
• 20 African Americans

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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
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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)

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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)
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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

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Patient Cohort
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Patient Ethnicity
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HAART Regimen
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PCR Methodology
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PCR Methodology
Step 2 Allele specific PCR (asPCR)
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DNA Sequencing
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Study samples

• 44 samples per batch, 3 ve controls (GG, GT,
  TT), 1 ve control (dH2O)

• 3 batches (136 samples) OK

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CYP2B6 G516T Frequency
TT 6
Plt0.05 (?2)
TT 16
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Questionnaire Analysis (1)
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Questionnaire Analysis (2)

• Questionnaire score vs HAART regimen (1)

P 0.67

• No significant difference in QS between HAART
  regimen groups

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Questionnaire Analysis (3)

• Questionnaire score vs HAART regimen (2)

ON EFV
OTHER
NONE

• No significant difference in QS between genotype
  groups when analysed according to HAART regimen

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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)

• No significant difference (P 0.63) found

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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

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