1. Circadian Genes, Stress Axis and Fetal Alcohol Spectrum Disorder
PI: Dipak K. Sarkar

2. Goal: to determine changes in levels of Per1 and Per2 gene methylation and expression and cortisol and testosterone from biological samples of FASD and non-FASD children from longitudinal studies conducted by CIFASD in order to identify a stable epigenetic signature of a specific group or all FASD patients.

3. Fetal alcohol exposed offspring often shows health problems
Fetal Alcohol Syndrome, partial
FAS or physical expression
Sleep-wake disturbance
Mental diseases such as anxiety and depression
Abnormality in coping stress
Metabolic disease
Immune dysfunction & Cancer

4. Studies in laboratory rodents
Fetal ethanol exposure alter the daily rhythm of clock genes and POMC levels in the adult hypothalamus of rats
Fetal alcohol exposed adult rats show abnormality in clock gene expression in the arcuate nucleus and in the SCN and in POMC expression in the arcuate nucleus. (Chen CP, et al J Neurochem. 97: ).

5. Studies in laboratory rodents
Fetal alcohol exposures induce hyper-response of stress hormones following stress in adult mice
Logan and Sarkar, unpublished

6. Studies in laboratory rodents
Per2 knocking down reduces beta-endorphin levels and prevents fetal alcohol-induced increase in stress-induced corticosterone release in mice
Agapito and Sarkar, Endocrinology, in revision

7. Does fetal alcohol exposure program the POMC system to control the neuroendocrine-immune axis functions?
Reward
Amygdala
N. Accumb.
SCN
(-)
CRH, ANS
PVN
-EP
(-)
Arcuate
POMC
PIT
ACTH
-EP
(+)
(+)
(-)
Lymphoid
organs
CORT
Adrenal
NK cells

8. Why fetal alcohol exposures alter Per2 and POMC gene expression
Why fetal alcohol exposures alter Per2 and POMC gene expression? Is there any role of the epigenetic mechanism?
Adopted from S. Pandey

9. Fetal alcohol’s epigenetic programming of POMC neurons
Microarray analysis revealed differential significant expression of 20 genes with a P<0.001 with a 2 fold change and around 2,000 genes with a P<0.01 and a P<0.05 with less than 2 fold change in linear scale. We linked particular sets of these 20 genes to biological functions including methylation, transcriptional and translational regulation, alcohol metabolism, folate metabolism and immunity (Table 2).
Bekdash, Agapito and Sarkar, unpublished

10. Fetal alcohol’s epigenetic programming of POMC neurons
Fetal Alcohol Increases Methylation States of Individual CpG Sites in the Hypothalamic POMC Promoter
Govorko et al., 2012, Biol Psychiatry

11. 5’-Aza or TSA FAE Pomc gene repression Pomc gene activation
SAM
HDACs
Dnmts Ac
Hypermethylation
MBDs
Deacetylation
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Inaccessible chromatin
Hypermethylated CpG island
SAM
Acetylation
HATs
Fig. 1. Conceptual diagram of the epigenetic effects of fetal alcohol exposure on POMC gene. Dnmt=DNA methyltransferase, MBD (methyl-binding domain proteins), HDAC=histone deacetylase, HAT= Histone acetyltransferase, Ac=acetylation, FAE=fetal alcohol exposure, TSA=trichostatin, 5’-Aza=5’-azacytidine, CH3=methyl group, SAM=S-adenosylmethionine.
Dnmts Ac
Normal methylation
CH3
CH3
CH3
CH3
Accessible chromatin
CpG island
Bekdash et al, in communication
FAE
Pomc gene activation

12. Fetal alcohol’s epigenetic programming of POMC neurons
Choline supplementation reversed ethanol effects on POMC gene promoter methylation, POMC gene expression, β-EP production, and normalized the corticosterone hyper-response to LPS challenge in alcohol-exposed rats.
Bekdash et al, ACER i2013

13. Fetal alcohol’s epigenetic programming of POMC neurons
Govorko et al.,2012, Biological Psychiatry
Rachdaoui and Sarkar, in press

14. Interrelationship between the epigenetic programming of POMC and Per2 genes: using fetal alcohol exposed rats
Logan and Sarkar, unpublished

15. Interrelationship between the epigenetic programming of Per2 genes and stress axis: using human alcoholic patients
30 light social drinking controls (HC) matched to 70 treatment engaged alcohol dependent (AD) individuals were used. Methylation of the Per2 DNA expression as assayed by MSRP in AD vs HC (P<0.002). Increased methylation levels were significantly associated with stress-induced cortisol (r=.39, P<0.03) and ACTH levels (r=.41, P<0.02). Methylation/unmethylation Ratio in AD vs HC (p<.002).
Sinha and Sarkar, unpublished

16. FAS and fetal alcohol exposed children show persistent hypermethylation of POMC gene
Collaborative work with Dr. Foroud and CIASD

17. FAS and fetal alcohol exposed children show persistent hypermethylation of Per2 gene
Collaborative work with Dr. Foroud and CIASD

18. Interrelation with Aims of the Consortium and Other Projects
We are working with other Consortium projects conducted by Drs. Tatiana Foroud, Christina Chamber, Elizabeth R. Sowell, Sara Mattson, Jeff Wozniak and Claire Coles.

19. Questions we are addressing and our expected outcome
Question 1. Do methylation and expression levels of Per genes correlate with the stress abnormality (high cortisol)?
Question 2. Can these methylation sites and patterns be used as biomarkers that predict future endophenotypes of stress abnormalities (e.g., anxiety, hyperactivity)?
Question 3. If Per genes hypermethylation is involved in stress abnormalities, whether this is manifested in patients with FASD?
Expected outcome: These studies may allow identification of a stable epigenetic marker for earlier detection of alcohol-related birth outcomes.