MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
DNA methylation as an epigenetic marker in HIV-2 disease in West
Africa?
Alberta DavisMRC Laboratories, Gambia18th January 2013
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
HIV pathogenicity and clinical outcomes
• AIDS: causal agents; retroviruses» HIV-1 » HIV-2
• HIV-1 “elite” controllers, long term non progressors (LTNPs), exposed uninfected. (Saksena et al 2007)
• HIV-2 infection is less progressive (low VL, low transmission, slow decline of CD4 T cells, prolonged survival).
• 20% of HIV-2 infected individuals exhibit high VL and a clinical presentation indistinguishable from AIDS in HIV-1. (Esbjörnsson et al, 2012)
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Determinants of progression to AIDS
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Host determinants
• CCR5 receptor:
• Viral entry into CD4 T cells.
• mutation confers resistance to infection with HIV.
• Variable CCR5 expression in HIV-1 is associated with matrices of infection and pathogenesis.
• Low and protective levels of CCR5 on CD4+ T cells is exhibited in HIV-2 infections (Shea et al 2004).
• IL-2 –CCR5 are coregulated genes and CCR5 can influence signaling
events during T cell activation (Camargo et al 2009).
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Epigenetics and its regulation of genes
• Epigenetic mechanisms: Heritable but reversible without change in DNA sequence
DNA methylation in CpG dinucleotide = gene repression
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
hypothesis
“ The DNA methylation status of regulatory regions of CCR5 and IL-2 serve as a determinant of differential HIV-2 pathogenicity”
• Objective » To determine the methylation status at CCR5 cis-
regulatory CpG sites and IL-2 gene loci in progressive and non progressive infections
» Evaluate the frequencies of CCR5 genetic variants
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Methods
• HIV-2 rich cohorts – Guinea Bissau and Gambia.
• Interrogated patient database based on previous studies of HIV-2 non progression (Berry et al 2002, Schim van der Loeff et al 2010).
• Categorised samples into various groups based on CD4 and Viral load
• Progressor: CD4 <200 cells/ul and VL > 10,000 copies/ml
• Non progressor: CD4 >500 cells/ul and VL < 100 copies/ml
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Overview of patient profiles
Demographic and virologic characteristics of 36 HIV-2 subjects n %Characteristic Categories/units Age: Median(range) years 45 (21-73)CD4: Median(range) counts 573 ( 10 - 2720)Viral load: Median(range) counts 22146 ( 100 - 607000) Sex Male 15 41.7 Female 21 58.3 Ethnicity Mandinka 13 36.1 Manjago 9 25 Other 14 38.8 CD4 counts Low (< 200 cells/ul) 15 41.7 High (> 500 cell/ul) 21 58.3 Viral load counts Low (< 100 copies/ml) 15 41.7 High (> 10,000 copies/ml) 21 58.3 CD4/VL H/H 9 25 H/L 12 33.3 L/H 12 33.3 L/L 3 8.3
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Specific CpG in CCR5 cis regulatory sites
Bisulphite modification and pyrosequencing (CCR5 and IL-2)
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Association between DNA methylation and CD4
CD4 > 500 (n = 21)
CD4 > 200 (n = 15)
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Correlation CD4 and methylation of CCR5 and IL-2
510
1520
2530
Met
hyla
tion
2 4 6 8Log(CD4)
n=36, rho=0.501 p value=0.002Methylation against CD4 at Distal Window
3040
5060
70M
ethy
latio
n
2 4 6 8Log(CD4)
n=36, rho=-0.454 p value=0.005Methylation against CD4 at IL-2
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Progressor (L/H) and non progressor (H/L) phenotypes show different methylation patterns
L/H (n = 12)
H/L (n = 12)
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Conclusion
• The CD4 count and VL load could be influenced by methylation levels.
• Viral control in non progressors is being achieved by low CCR5 expression and maintenance of CD4+ T cells which produce IL-2.
• Progressors are less methylated at CCR5 regulatory regions
but more methylated at the IL-2 locus than non progressors.
• No significant difference was found at CCR5 promoter 2 possibly because Pr2 is active only upon T cell activation
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Recruitment of patient and blood sampling in Guinea Bissau
Duration in the cohort (1989, 1997, 2003, 2006)
• HIV-2 non progressor,HIV-2 progressor,HIV-1 asymptomatics, Healthy controls.
• CCR5 and IL-2 DNA methylation • CCR5 and IL-2 mRNA expression levels • CCR5 allelic discrimination• Immunophenotyping by flow cytometry• Multiplex cytokine analysis by Bioplex-cytokine analysis
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Acknowledgements
UTHSCSA
Prof Sunil K. Ahuja Dr He Weijing
Komathy JayasekarUna AluyenShivali Chag
MRC Unit
Dr Assan Jaye
Pa Saidou Chaw, MD Dr Alfred NgwaRamou-Sarge NjieGilleh Thomas
James Jafali
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
THANK YOU!