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

Clock gene PER2 and stress-regulatory gene POMC are targets of alcohol Nat Med. 2005 Jan;11(1):35-42. The clock gene Per2 influences the glutamatergic system and modulates alcohol consumption. Spanagel R, Pendyala G, Abarca C, Zghoul T, Sanchis-Segura C, Magnone MC, Lascorz J, Depner M, Holzberg D, Soyka M, Schreiber S, Matsuda F, Lathrop M, Schumann G, Albrecht U. Ups J Med Sci. 2010 Feb;115(1):41-8. The clock gene PER2 and sleep problems: association with alcohol consumption among Swedish adolescents. Comasco E, Nordquist N, Göktürk C, Aslund C, Hallman J, Oreland L, Nilsson KW. PLoS One. 2013;8(3):e59136 Association of PER2 genotype and stressful life events with alcohol drinking in young adults. Blomeyer D, Buchmann AF, Lascorz J, Zimmermann US, Esser G, Desrivieres S, Schmidt MH, Banaschewski T, Schumann G, Laucht M. Neuroreport. 2012 Jan 25;23(2):98-102. Chronic stress affects PERIOD2 expression through glycogen synthase kinase-3β phosphorylation in the central clock. Kinoshita C, Miyazaki K, Ishida N. J Neural Transm. 2010 Apr;117(4):513-9. DNA methylation of the POMC gene promoter is associated with craving in alcohol dependence. Muschler MA, Hillemacher T, Kraus C, Kornhuber J, Bleich S, Frieling H.

Clock gene PER2 and stress-regulatory gene POMC knocking down increases anxiety-like behaviors and stress hyper-responsiveness Eur J Neurosci. 2013 Jan;37(2):242-50. Circadian genes Period 1 and Period 2 in the nucleus accumbens regulate anxiety-related behavior. Spencer S, Falcon E, Kumar J, Krishnan V, Mukherjee S, Birnbaum SG, McClung CA. Endocrinology. 2009 May;150(5):2153-60. The role of mPer2 clock gene in glucocorticoid and feeding rhythms. Yang S, Liu A, Weidenhammer A, Cooksey RC, McClain D, Kim MK, Aguilera G, Abel ED, Chung JH. Psychopharmacology (Berl). 2008 Sep;200(1):105-15. Influence of beta-Endorphin on anxious behavior in mice: interaction with EtOH. Grisel JE, Bartels JL, Allen SA, Turgeon VL. Psychoneuroendocrinology. 2008 May;33(4):425-36. Control of hormonal stress reactivity by the endogenous opioid system. Bilkei-Gorzo A, Racz I, Michel K, Mauer D, Zimmer A, Klingmüller D, Zimmer A.

Hypothesis Chronic alcohol drinking produces epigenetic marks on PER2 and POMC genes to manifest stress disorders and hence methylation changes in PER2 and POMC genes in alcoholic women could potentially identify the birth outcome of their FASD child.

FAS and fetal alcohol exposed children show persistent hypermethylation of POMC gene Study using salivary DNA and MSP Collaborative work with Drs. Tatiana Foroud, Omkaram Gangisetty, and CIFASD

Fetal alcohol causes hypermethylation of CpG islands at the POMC promoter Study using salivary DNA and pyrosequencing Collaborative work with Drs. Tatiana Foroud, Lourdes Serrano, Omkaram Gangisetty, and CIFASD

FAS and fetal alcohol exposed children show persistent hypermethylation of PER2 gene Study using salivary DNA and MSP Collaborative work with Drs. Tatiana Foroud, Omkaram Gangisetty and CIFASD

Mother smoking habits do not influence fetal alcohol increased methylation of POMC and PER2 genes Study using salivary DNA and MSP Collaborative work with Drs. Tatiana Foroud, Omkaram Gangisetty and CIFASD

FAS and fetal alcohol exposed children with or without nicotine exposures show no changes in PER1 gene methylation Study using salivary DNA and MSP Collaborative work with Drs. Tatiana Foroud, Omkaram Gangisetty and CIFASD

Increased POMC gene methylation is assoicated with reduced POMC expression in alcoholic patients Study using blood DNA, RNA, MSP and RT-PCR Collaborative work with Drs. Rajita Sinha and Omkaram Gangisetty

Increased hypermethylation of CpG islands at the POMC promoter is also observed in alcoholic patients Study using blood DNA and pyrosequencing Collaborative work with Drs. Rajita Sinha and Omkaram Gangisetty

Increased PER2 gene methylation is assoicated with reduced PER2 expression in alcoholic patients Study using blood DNA, RNA, MSP and RT-PCR Collaborative work with Drs. Rajita Sinha and Omkaram Gangisetty

Increased POMC and PER2 gene methylation are assoicated with reduced POMC and PER2 expression in animal model of FASD Study using hypothalamic DNA and RNA Collaborative work with Dr. Demitry Govorko

Fetal alcohol exposed children show increased basal beta-endorphin levels Study using saliva and ELISA Collaborative work with Drs. Jeffrey Wozniak, Kristina Urban, Elizabeth Sowell, and Shaima Jabar, and CIFASD

Fetal alcohol exposed children show increased basal cortisol levels Study using saliva and ELISA Collaborative work with Drs. Jeffrey Wozniak, Kristina Urban, Elizabeth Sowell, and Shaima Jabar, and CIFASD

A positive correlation exists between PER2 gene methylation and cortisol in alcoholic patients Study using blood DNA and plasma Collaborative work with Drs. Rajita Sinha and Omkaram Gangisetty

Reduced POMC and PER2 expression is associated with increased basal corticosterone and corticosterone response to stress in animal model of FASD Study using hypothalamic DNA and RNA Collaborative work with Dr. Maria Agapito and Ryan Logan

Choline supplementation prevented alcohol epigenetic marks on POMC gene and stress axis in animal model of FASD Study using hypothalamic DNA, RNA and plasma Bekdash et al., ACER 2014

Choline supplementation may have beneficial effects in suppressing alcohol epigenetic marks on POMC gene in FAS patients Study using PBMC and MPR Collaborative work with Drs. Jeffrey Wozniak and Omkaram Gangisetty

Choline supplementation may have beneficial effects in suppressing alcohol epigenetic marks on PER2 gene in FAS patients Study using PBMC and MPR Collaborative work with Drs. Jeffrey Wozniak and Omkaram Gangisetty

Summary and Conclusions 1. Chronic alcohol consumptions epigenetically modify PER2 and POMC genes to suppress the expression of these genes leading to stress hyper-responsiveness and anxiety-like behaviors. 2. Alcohol epigenetic marks on PER2 and POMC genes and the stress axis may transmit to offspring and thereby measuring there levels in biological fluids of alcoholic pregnant women could potentially identify the birth outcome of FASD child. 3. Choline supplementation can reverse some of these epigenetic marks and thereby may have beneficial effects on stress control mechanism in FASD patients.

A. Drinkers with FAS child B: Drinkers with unaffected child Ongoing projects 1. Measuring PER2 and POMC methylation levels in three group of patient blood DNA samples: A. Drinkers with FAS child B: Drinkers with unaffected child C: Non drinkers 2. Measuring PER2 proteins profiles in salivary samples of FAS and control children 3. Writing a manuscript for publication

Future goals Establish collaboration with CIFASD investigators to submit a grant proposal to determine: 1. FASD signature marks on epigenome in both FAS/non-FAS children and mothers (blood, saliva or cord blood) who gave birth to FAS/non-FAS children using MeDIP and bisulfite sequencing (problem of alignment accuracy to repetitive regions in the genome will result in less accurate analysis of methylation in those regions). Study whether the epimutations is common between mother and children using pyrosequencing assays at single nucleotide resolution. 2. Identify the biological pathways (ChIP sequencing and RNA sequencing) affected in FAS from pathway analysis and correlate them with behavioral (e.g., hyperactivity disorders) and health outcomes (e.g., immune health). 3. Whether epigenetic modifiers have any beneficial effects in prevention of behavioral and health abnormalities in FASD patients?