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Thyroid hormone (TH) affects virtually all cells and tissues in the human body. The major biologically active TH is T3, and its genomic actions are mediated by binding to nuclear T3 receptors (TRs) that regulate transcription of target genes (1). Several receptor isoforms are encoded by the THRA and THRB genes of which TRα1, TRβ1, and TRβ2 are the truly T3 binding isoforms (2). The TR isoforms have a distinct expression pattern, with a predominance of TRα1 in brain, heart, and bone and TRβ1 in the liver, kidney, and thyroid. TRβ2 is mainly expressed in the hypothalamus and pituitary and is therefore involved in the regulation of the hypothalamus-pituitary-thyroid axis (3).Despite the classical clinical features resulting from insufficient or excessive TH levels that have been recognized for more than 100 years, the underlying molecular mechanisms in humans are not well understood. Knowledge of gene expression modulated by TH is largely derived from animal models or in vitro cellular studies used to explore which genes are regulated by TH (4–7). Expanding knowledge of the effects of TH on gene expression in human tissues will provide more insight in the molecular basis of TH action and may lead to a better understanding of the clinical effects of TH in humans.Progress is limited because most human tissues are not easily accessible. However, blood can be regarded as circulating tissue and contains various cell types including erythrocytes, leukocytes, and platelets (8). RNA in whole blood is largely determined by leukocytes. Peripheral blood mononuclear cells (lymphocytes and monocytes) have been shown to mainly express the TRα isoform (9). Therefore, analysis of gene expression in whole blood may potentially be used as proxy for other TRα-expressing tissues. To study the effects of TH on gene expression in human TRα-expressing cells, we performed next-generation RNA sequencing (RNA-seq) in whole blood cells from athyroid patients off and on levothyroxine (LT4) treatment.Patients were recruited via the outpatient clinic of the Erasmus Medical Center, which is a tertiary referral center for differentiated thyroid cancer (DTC). Patients with DTC undergoing thyrotropin (TSH)-stimulated 131I therapy after withdrawal of LT4 were asked to participate in the study. Patients were eligible for inclusion if they had no other malignancies or an active inflammatory disease, were not using any drugs known to influence TH metabolism, and were between 18 and 80 years of age. A discovery and a validation cohort were created according to the same protocol.The study protocol was approved by the Medical Ethics Committee of the Erasmus Medical Center (MEC 2012-561). Written informed consent was obtained from all study participants.Peripheral blood samples were obtained from all participants when overt biochemical hypothyroidism was achieved by withdrawal of LT4 substitution in thyroidectomized patients and when TSH suppression was achieved after restarting LT4 replacement therapy. Serum free T4 (reference range, 11 to 25 pmol/L), total T4 (reference range, 58 to 128 nmol/L), and total T3 (reference range, 1.4 to 2.5 nmol/L) concentrations were measured by chemoluminescence assays (Vitros ECI Immunodiagnostic System; Ortho-Clinical Diagnostics, Rochester, MI). Serum TSH (reference range, 0.4-4.3 mU/L) was measured by an immunometric assay (Immulite 2000 XPi; Siemens, Den Haag, the Netherlands). Whole blood samples were collected in PAXgene tubes (Hombrechtikon, Switzerland). PAXgene tubes contain a proprietary reagent that immediately stabilizes intracellular messenger RNA (mRNA), thus reducing mRNA degradation and inhibiting gene induction after phlebotomy.