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NIH Public Access Author Manuscript Psychiatry Res. Author manuscript; available in PMC 2014 November 30. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Psychiatry Res. 2013 November 30; 210(1): 287–293. doi:10.1016/j.psychres.2013.05.026. Assessment of gene expression in peripheral blood using RNAseq before and after weight restoration in anorexia nervosa Yunjung Kima,1, Sara Elizabeth Traceb,1, James Joseph Crowleya, Kimberly Ann Brownleyb, Robert Mark Hamerb,c, David Stephen Pisetskyd,e,f, Patrick Francis Sullivana,b, and Cynthia Marie Bulikb,g,* aDepartment of Genetics, University of North Carolina at Chapel Hill, NC, USA bDepartment of Psychiatry, University of North Carolina, Chapel Hill, NC, USA cDepartment of Biostatistics, University of North Carolina, Chapel Hill, NC, USA dDepartment of Medicine, Duke University, Durham, NC, USA eDepartment of Immunology, Duke University, Durham, NC, USA fMedical Research Service, Durham Veterans Administration Medical Center, Durham, NC, USA gDepartment of Nutrition, University of North Carolina, Chapel Hill, NC, USA Abstract We examined gene expression in the blood of six females with anorexia nervosa (AN) before and after weight restoration using RNAseq. AN cases (aged 19-39) completed clinical assessments and had blood drawn for RNA at hospital admission (T1, < ~75% ideal body weight, IBW) and again at discharge (T2, ≥ ~85% IBW). To examine the relationship between weight restoration and differential gene expression, normalized gene expression levels were analyzed using a paired design. We found 564 genes whose expression was nominally significantly different following weight restoration (p < 0.01, 231 increased and 333 decreased). With a more stringent significance threshold (false discovery rate q < 0.05), 67 genes met criteria for differential expression. Of the top 20 genes, CYP11A1, C16orf11, LINC00235, and CPA3 were down-regulated more than two- fold after weight restoration while multiple olfactory receptor genes (OR52J3, OR51L1, OR51A4, OR51A2) were up-regulated more than two-fold after weight restoration. Pathway analysis revealed up-regulation of two broad pathways with largely overlapping genes, one related to protein secretion and signaling and the other associated with defense response to bacterial regulation. Although results are preliminary secondary to a small sample size, these data provide initial evidence of transcriptional alterations during weight restoration in AN. © 2013 Elsevier Ireland Ltd. All rights reserved. *Corresponding author at: University of North Carolina, Psychiatry, CB #7160, Chapel Hill, NC 27599, United States. Tel.: +1 919 843 1689; fax: +1 919 843 8802. [email protected]. 1These authors contributed equally to this work. Financial Disclosures. Dr. Sullivan was on the SAB of Expression Analysis (Durham, NC). The other authors report no biomedical financial interests or potential conflicts of interest. Publisher©s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Kim et al. Page 2 Keywords NIH-PA Author Manuscript NIH-PA Author Manuscriptbiomarkers; renourishment; NIH-PA Author Manuscript transcriptome; eating disorder; genetic; genome 1. Introduction Anorexia nervosa (AN), a severe psychiatric illness marked by extremely low body weight, fear of weight gain, and inability to recognize the seriousness of the low weight, carries the highest mortality rate of any psychiatric disorder (Sullivan 1995; Zipfel et al., 2000; Birmingham et al., 2005; Millar et al., 2005; Papadopoulos et al., 2009). The DSM-IV-TR (American Psychiatric Association, 2000) differentiates two types of AN; the restricting type (AN-R) and the binge-purge type (AN-BP). Unlike individuals with AN-R, individuals with AN-BP regularly engage in binge eating and/or compensatory behaviors. As in other forms of starvation, AN is associated with biochemical, metabolic, immunologic, and sensory abnormalities (Mira et al., 1987; Umeki 1988; Nova et al., 2002; Mont and Castro, 2003; Millar et al., 2005; Ulger et al., 2006). Individuals with AN who are less than 75% ideal body weight (IBW) are typically hospitalized for medically supervised weight restoration (American Psychiatric Association, 2000). Inpatient treatment is costly (Krauth et al., 2002) and relapse is common (Carte et al., 2004). There are no robust biological indices of risk, illness severity, or treatment response, and the identification of such biomarkers is an urgent area of inquiry. To be clinically useful, AN biomarkers must distinguish indices of starvation from indices of disease. Indeed, a number of metabolic, endocrine, and neural biomarkers attributed to malnutrition have been found in AN (e.g., alterations in neuropeptide Y, leptin, ghrelin, orexin A, corticotrophin-releasing hormone, cholecystokinin, pancreatic polypeptide beta- endorphin, and brain derived neurotrophic factor; Lob et al., 2003; Nakazato et al., 2003; Connan et al., 2007; Støving et al., 2009; Bronsky et al., 2011). Other biomarkers that have been reported to persist following renourishment in AN, and thus could represent an index of disease status, include abnormalities of the dopamine (Kaye et al., 1999; Bergen et al., 2005) and serotonin (Bailer et al., 2005; Galusca et al., 2008) neurotransmitter systems. The majority of such studies, however, tested a single candidate biomarker that is a part of a metabolic or neurochemical system believed to be related to the etiology of AN. This candidate approach has been unsuccessful in the vast majority of complex biomedical traits, suggesting that a more global and unbiased search is warranted. Although the number of studies is limited, changes in the expression of candidate genes have been reported in individuals with AN (Frieling et al., 2008; Ehrlich et al., 2010). Ehrlich et al. (2010) used quantitative polymerase chain reaction (PCR) to examine proopiomelanocortin (POMC) splice variant levels in peripheral blood ribonucleic acid (RNA). The long POMC splice variant was higher in underweight women with AN than weight-recovered individuals and healthy controls. Alterations in POMC expression were interpreted to be the result of malnutrition, rather than a persisting trait marker of AN. Similarly, Kahl et al. (2004) examined expression of tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), interleukin-6 (IL-6), and interleukin-10 (IL-10). An increase in TNF-α and IL-6 expression was found in individuals with AN at hospital admission compared with controls, and the expression of TNF-α remained significantly higher in those with AN who were weight restored, while IL-6 expression decreased. The authors concluded that TNF-α may contribute to metabolic abnormalities in AN even following weight restoration. Janas- Kozik et al. (2008) found that a leptin receptor transcript showed statistically significant differences between individuals with AN and controls. Several other investigations compared expression of P-glycoprotein (Storch et al., 2008) and prohormone Psychiatry Res. Author manuscript; available in PMC 2014 November 30. Kim et al. Page 3 preproenkephalin (Weiss et al., 2010), failed to find significant differences. Although limited in number, these studies have provided some preliminary information regarding NIH-PA Author Manuscript NIH-PA Author Manuscripttranscriptomic NIH-PA Author Manuscript profiling in AN. However, these investigations have been hampered by the study of small numbers of candidate genes and a lack of within-subjects longitudinal data. Whole transcriptome expression profiling is a powerful, unbiased method capable of identifying genes and biological pathways correlated with a phenotypic trait or environmental perturbation. As such, gene expression levels can serve as a biomarker for a biomedically relevant state. Whole blood is an attractive tissue source for the identification of gene expression biomarkers related to AN for two main reasons. First, blood is easily accessible whereas the target organ of interest (brain) is not. Second, genes expression levels in peripheral blood are well correlated with multiple central nervous system tissues (median non-parametric correlation of 0.5; Sullivan et al., 2006), suggesting cross-tissue relevance for expression changes. An important next step towards generating candidate biomarkers for further exploration in AN is to determine how transcriptome expression patterns change as patients gain weight. Given the profound physiological changes that occur with starvation and renourishment, as well as numerous reports from the animal literature citing transcriptomic changes secondary to starvation and caloric restriction (Narnaware and Peter, 2001; Drew et al., 2008; Dhahbi et al., 2012; Mitchell et al., 2012; Plank et al., 2012), we expected to see transcriptomic changes during renourishment in AN. Although weight gain is not the only index of recovery, it is an essential first step in treatment, and identifying differential expression between the acutely underweight and weight restored state is an essential component of our ultimate