Rachel Yehuda, Nikolaos P. Daskalakis, Linda M. Bierer, Heather N

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Holocaust Exposure Induced Intergenerational Effects on FKBP5 Methylation  Rachel Yehuda, Nikolaos P. Daskalakis, Linda M. Bierer, Heather N. Bader, Torsten Klengel, Florian Holsboer, Elisabeth B. Binder  Biological Psychiatry  Volume 80, Issue 5, Pages 372-380 (September 2016) DOI: 10.1016/j.biopsych.2015.08.005 Copyright © 2016 Terms and Conditions

Figure 1 Schematic representation of the human FKBP5 locus with intron 7 glucocorticoid receptor binding sequence investigated in this study. The upper panel depicts the FKBP5 locus in 5’-3’ orientation. Black bars represent the 11 exons. The transcription start site is highlighted in green. The lower panel represents the intron 7 amplicon (476 base pair) chosen for DNA methylation analysis (primer sequence dark blue/italicized). Since pyrosequencing can only reliably generate short reads, the six cytosine-phosphate-guanine (CpG) sites (red) analyzed in three bins based on the proximity to three consensus glucocorticoid response elements (GREs) are represented in bold/underlined [pyrosequencing primers are described in Klengel et al. (38)]. The two CpGs of bin 1 were upstream of all GREs, the three CpGs of bin 2 are surrounding the first GRE, and bin 3 represents the CpG within the third GRE. The chromosomal position (hg19) of the CpG sites is indicated on the left and the right of the lower panel. SNP, single nucleotide polymorphism. Biological Psychiatry 2016 80, 372-380DOI: (10.1016/j.biopsych.2015.08.005) Copyright © 2016 Terms and Conditions

Figure 2 Methylation at FKBP5 intron 7, bins 1, 2, and 3 for Holocaust survivors (A), Holocaust survivor offspring (B), and their respective comparison subjects. The percent methylation (mean ± SEM) is represented by red bars for Holocaust survivor parents (F0) and their offspring (F1) (F0: n = 32, F1: n = 22) and by white bars for F0 and F1 control subjects (F0: n = 8, F1: n = 9). Division of sites into bins is indicated. ⁎p < .05. Biological Psychiatry 2016 80, 372-380DOI: (10.1016/j.biopsych.2015.08.005) Copyright © 2016 Terms and Conditions

Figure 3 Relationship between FKBP5 intron 7, site 3 percent methylation and Childhood Trauma Questionnaire physical abuse subscale score. The carriers of the FKBP5 rs1360780 risk allele (n = 18) are depicted by black squares and the carriers of the protective genotype (n = 13) are depicted by black open circles. Significance was set at p < .05. F1, first generation offspring. Biological Psychiatry 2016 80, 372-380DOI: (10.1016/j.biopsych.2015.08.005) Copyright © 2016 Terms and Conditions

Figure 4 Relationship between original parent generation (F0) and first generation (F1) FKBP5 intron 7 bin 3/site 6 percent methylation. Parent-offspring pairs are represented by red squares for Holocaust survivors (n = 23) and by blue open circles for control subjects (n = 10). Significance was set at p < .05. Biological Psychiatry 2016 80, 372-380DOI: (10.1016/j.biopsych.2015.08.005) Copyright © 2016 Terms and Conditions

Figure 5 Relationship between FKBP5 intron 7, bin average percent methylation, and wake-up salivary cortisol (ng/dL) in first generation (F1). Holocaust survivor offspring (n = 14) are depicted by red squares and control subjects (n = 8) are depicted by blue open circles. Significance was set at p < .05. Biological Psychiatry 2016 80, 372-380DOI: (10.1016/j.biopsych.2015.08.005) Copyright © 2016 Terms and Conditions