MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world DNA methylation as an epigenetic marker in.

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)

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Determinants of progression to AIDS

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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

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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

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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

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Specific CpG in CCR5 cis regulatory sites

Bisulphite modification and pyrosequencing (CCR5 and IL-2)

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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)

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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

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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!