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

Goal: to determine changes in levels of Per1 and Per2 gene methylation and expression and cortisol and ACTH 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 or all FASD patients.

Why clock genes? Children with FASD have significantly more sleep disturbances than typically developing children, including increased bedtime resistance, shortened sleep duration, increased sleep anxiety, and increased night awakenings and parasomnias (Wengel et al., 2011). Many children diagnosed with FASD have long-standing sleep disturbances and behavioral (Jan et al., 2010) and immune problems (Johnson, 1981). Research has consistently linked sleep problems to emotional and behavioral problems in adolescents (Roberts et al., 2008). Animal studies also linked sleep problems with immune abnormalities and higher incidence of cancer (Logan and Sarkar, 2012) Clock gene variation is associated with sleep problems among adolescent boys (Comasco et al., 2010) Data show that children with FASD have multiple sleep problems, significantly more sleep disturbances than typically developing children, including increased bedtime resistance, shortened sleep duration, increased sleep anxiety, and increased night awakenings and parasomnias. Images from: http://images.search.yahoo.com

Why clock genes? Studies conducted in rats suggest that developmental alcohol exposure may indeed interfere with circadian clock function as evidenced by a shortened circadian sleep-wake cycle and changes in the release of certain brain chemicals by SCN cells in the hypothalamus (Earnest et al., 2006). Studies conducted in rodents also suggest that prenatal alcohol exposures reduces expression of clock genes in the brain (Chen et al., 2006). Images from: http://images.search.yahoo.com

What are clock genes? Images from: http://images.search.yahoo.com Alcoholism and FASD?? Images from: http://images.search.yahoo.com

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. 2006 J Neurochem. 97:1026-1033).

Fetal alcohol exposures induce stress hyper-response and increase alcohol drinking behaviors following stress in adult mice Logan and Sarkar, unpublished

Studies in laboratory rodents Fetal ethanol exposure alter the daily rhythm of cytolytic factors and cytokines levels in the spleen. Arjona A, Boyadjieva N, Sarkar DK. 2006 Alcohol Clin Exp Res. 30:1039-1044.

Studies in laboratory rodents Alcohol exposure in utero enhances prostate tumorigenesis in rats Mojtehedzadeh et al. in communication

Studies in laboratory rodents Alcohol exposure in utero enhances the incidence of mammary adenocarcinoma following MNU treatment in rats Normal Hyperplasia Incidence of MNU-induced tumors Adenoma Adenocarcinoma Polanco et al., ACER (2010)

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, Logan and Sarkar, unpublished

Studies in laboratory rodents THE CIRCADIAN MOLECULAR CLOCK REGULATES THE EXPRESSION OF CYTOLYTIC FACTORS AND CYTOKINES Effect of Per2 knockdown on the levels of cytolytic factors and cytokines in NK cells Arjona A, Sarkar DK. 2006. Brain, Behavior, and Immunity. 20-469-472.

Per2 deletion increases the incidence of cancer Studies in laboratory rodents Per2 deletion increases the incidence of cancer Lee CC. Tumor suppression by the mammalian Period genes. Cancer Causes Control. 2006 May;17(4):525-30.

Clock gene regulation of neuroendocrine-immune function Studies in laboratory rodents Clock gene regulation of neuroendocrine-immune function Logan and Sarkar, unpublished

Preliminary results (in human) 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

Hypothesis: alcohol’s modulating effect on DNA methylation makes an epigenetic mark on Per genes that serves to activate the HPA axis via suppression of the POMC gene this facilitates maladaptation of the HPA system. To test this hypothesis, we propose to measure levels of Per gene methylation and expression and stress hormones using the DNA, RNA and plasma samples of FASD and non-FASD patients available in CIFASD consortium.

Phenotypes (similar age) Sex (number) Longitudinal (years) We plan to determine whether Per1 and Per2 gene methylation changes in FASD patients.  Longitudinal studies need to be conducted  to assess if the epigenetic modification is stable or there is any change due to treatment and/or age.   For this we need to have the following if possible: Phenotypes (similar age) Sex (number) Longitudinal (years)   FAS Males  (N >12) Females (N >12) Year 1 Year 2 Year 3 FASD Males (N >12) Females (N >12) Year 1 Year 2 Year 3 Control Males (N >12) Females (N >12) Year 1 Year 2 Year 3 1. We will first measure Per1 and 2 methylation using methylation specific PCR (MSP) to quantitate the changes in gene methylation followed by Pyrosequencing Analysis for detecting the site of methylation. For MSP assays we will use about 1 microgram of DNA and for Pyrosequencing we will need about 2 microgram DNA.   2. We also want to determine whether Per1/2 gene methylation correlates with gene expression.  For this we would need 1 microgram RNA samples from the same patients. 3. I also propose to correlate per gene abnormalities with stress problems.  For this we would need plasma or saliva samples from the same patients.  I do not know whether you can also provide about 50 microliter plasma from these patients.

Progress with the human study Obtained approval by Rutgers (IRB exemption) to conduct study using de-identified human samples. 2. Signed a MTA for releasing CIFASD samples from IU to our facility We will be soon receiving salivary DNA samples from patients with FAS (female = 6, male = 7), alcohol exposed (female = 8, male = 14) and control (male =30, female – 18).

Questions to be addressed and 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.