Assessment of gene expression in peripheral blood using RNAseq before and after weight restoration in anorexia nervosa
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 (Carter 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 preproenkephalin (Weiss et al., 2010), failed to find significant differences. Although limited in number, these studies have provided some preliminary information regarding transcriptomic 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 toward 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 goal to differentiate biomarkers of starvation from biomarkers of disease. Thus, the purpose of this proof-of-concept investigation was to determine whether RNA-sequencing (RNAseq) interrogation of the peripheral blood transcriptome in AN can identify genes that differ before and after weight restoration. Specifically, this investigation tested for differential gene expression in the same individuals with AN at T1 (<~75% IBW) and T2 (≥ ~ 85% IBW).
Section snippets
Participants
Full methods and participant information are included in the Supplementary material (Table S1). Briefly, the participants were six females ages 19–39 who met DSM-IV-TR (American Psychiatric Association, 2000) criteria for AN and were admitted for inpatient treatment. Diagnosis was verified with the Structured Clinical Interview for DSM-IV (SCID-I/P; First et al., 2002). Five participants met criteria for AN-R type and one participant met criteria for AN-BP type. This study was approved by the
Laboratory
Results for the CBC and comprehensive metabolic panel for each participant at T1 and T2 can be found in Table S2. Laboratory values revealed limited abnormalities with no clear pattern across T1 and T2. Three individuals had a low white blood cell (WBC) count at T1 and two of these individuals had a low WBC count at T2. Four and three individuals had low absolute lymphocytes at T1 and T2, respectively. Only one individual had an elevated erythrocyte sedimentation rate (ESR) at T1 and her ESR
Discussion
Eight genes were up or down-regulated more than two-fold after renourishment in these patients with AN. Laboratory results revealed limited abnormalities that likely had minimal influence on differences in gene expression between T1 and T2. Expression of both up and down-regulated genes was only weakly correlated with changes in weight status, suggesting that subsequent studies with larger samples sizes may have the ability to detect biomarkers of illness rather than biomarkers that simply
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.
Acknowledgments
This project was supported by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Award no. UL1TR000083. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Additional funding was from the Foundation of Hope, Raleigh, NC. Dr. Trace was supported by National Institute of Health grant T32MH076694 (PI: Bulik) and 2012–2015 Hilda and
References (68)
- et al.
Gustatory and olfactory sensitivity in patients with anorexia and bulimia in the course of treatment
Journal of Psychiatric Research
(2008) - et al.
Persistent amenorrhoea in weight-recovered anorexics: psychological and biological aspects
Psychiatry Research
(2003) - et al.
Association between olfactory receptor genes, eating behavior and adiposity: results from the Quebec Family Study
Physiology and Behavior
(2012) - et al.
An investigation of hypothalamic-pituitary-adrenal axis hyperactivity in anorexia nervosa: the role of CRH and AVP
Journal of Psychiatric Research
(2007) - et al.
Brain hypometabolism of glucose in anorexia nervosa: normalizaiton after weight gain
Biological Psychiatry
(1996) - et al.
Promoter specific DNA methylation and gene expression of POMC in acutely underweight and recovered patients with anorexia nervosa
Journal of Psychiatric Research
(2010) - et al.
Eating disorders
Lancet
(2003) - et al.
Organic background of restrictive-type anorexia nervosa suggested by increased serotonin 1A receptor binding in right frontotemporal cortex of both lean and recovered patients: [18F]MPPF PET scan study
Biological Psychiatry
(2008) - et al.
Intact sensory function in anorexia nervosa
American Journal of Clinical Nutrition
(2012) - et al.
Origin and evolution of X chromosome inactivation
Current Opinion in Cell Biology
(2012)
Cytokine mRNA expression patterns in the disease course of female adolescents with anorexia nervosa
Psychoneuroendocrinology
Altered dopamine activity after recovery from restricting-type anorexia nervosa
Neuropsychopharmacology
Gene transcripts associated with BMI in the motor cortex and caudate nucleus of calorie restricted rhesus monkeys
Genomics
Anorexia nervosa: a disease with potentially lethal repercussions on the heart
Revista Española de Cardiología
Decreased levels of serum brain-derived neurotrophic factor in female patients with eating disorders
Biological Psychiatry
Effects of food deprivation and refeeding on neuropeptide Y (NPY) mRNA levels in goldfish
Comparative Biochemistry and Physiology. Part B, Biochemistry and Molecular Biology
Leptin, ghrelin, and endocannabinoids: potential therapeutic targets in anorexia nervosa
Journal of Psychiatric Research
Progesterone synthesis by the human placenta
Placenta
The endocrinopathies of anorexia nervosa
Endocrine Practice
Long-term prognosis in anorexia nervosa: lessons from a 21-year follow-up study
Lancet
Diagnostic and Statistical Manual of Mental Disorders
Altered brain serotioni 5-HT1A receptor binding after recovery from anorexia nervosa measured by positron emission tomography and [carbonyl11C]WAY-100635
Archives of General Psychiatry
Association of multiple DRD2 polymorphisms with anorexia nervosa
Neuropsychopharmacology
The mortality rate from anorexia nervosa
International Journal of Eating Disorders
Changes of orexin A plasma levels in girls with anorexia nervosa during eight weeks of realimentation
International Journal of Eating Disorders
Bacterial infections in anorexia nervosa: delayed recognition increases complications
International Journal of Eating Disorders
Relapse in anorexia nervosa: a survival analysis
Psychological Medicine
Differential regulation of the human CYP11A1 promoter in mouse brain and adrenals
Journal of Cellular Physiology
Database for annotation, visualization, and integrated discovery
Genome Biology
mRNA-Seq reveals complex patterns of gene regulation and expression in the mouse skeletal muscle transcriptome associated with calorie restriction
Physiological Genomics
Effect of starvation on transcriptomes of brain and liver in adult female zebrafish (Danio rerio)
Olfactory dysfunction in anorexia and bulimia
International Journal of Eating Disorders
Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I), Clinican Version
Epigenetic downregulation of atrial natriuretic peptide but not vasopressin mRNA expression in females with eating disorders is related to impulsivity
Neuropsychopharmacology
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These authors contributed equally to this work.