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Interaction Network Prediction and Analysis of Anorexia Nervosa.

Objectives
Anorexia Nervosa (AN) as a mental condition is a common eating disorder among young women. We aimed to shed lights on molecular behavior of this serious disorder in terms of protein interacting profile to provide further insight about its complexity.


Materials & Methods
The AN related genes were extracted from STRING database and included in interactome via Cytoscape software. The central nodes of the network were enriched via gene ontology (GO) by ClueGO+CluePedia and the action relationship between the nodes were determined by CluePedia.


Results
Six genes including LEP, INS, POMC, GCG, SST, and ALB were introduced as hub-bottlenecks that among them LEP, INS, and POMC were the super hub-bottlenecks based on further analysis. Action map analysis showed prominent role of hubs relative to bottlenecks in the network. Regulation of behavior, regulation of carbohydrate biosynthetic process, and regulation of appetite are the top associated processes for the identified hub genes.


Conclusion
Topological analysis proposed the five hub-bottlenecks as the most central genes in the network, these genes and their contributing biological terms may suggest additional importance in AN pathogenesis and thereby possible candidates for therapeutic usage. However, further studies are required to justify these findings.


Introduction
The eating disorder, anorexia nervosa (AN) accounts for the highest rate of death among the other psychiatric disorders (1). Its name became common from the late 19 th century. About 0.9% and 0.3% of women and men in the USA, respectively are diagnosed with AN. Patients with this disorder have an altered body shape since they fear to gain weight, and therefore not eating properly. About 90% of AN cases are women (2). In addition, the rate of this disorder is increasing in young women with unidentified etiology (3,4).
Moreover, the management of this heterogeneous eating disorder is allied with complications (5) as it shows resistance to the available treatments (6). In this regard, one of the important changes in AN patients is hormonal level alterations that can induce low bone mineral density (BMD) with irreversible consequences even after weight restoration (7).
The interaction between genetic and environmental factors are responsible for this disorder (8). Most of the molecular studies on AN, are limited to genetic, genome-wide and transcriptome studies.
The corresponding genetic and genome-wide studies are very heterogeneous and no candidate genes showed significantly replicated and related.
Additionally, none of the genes was significant at expression levels (9). A recent proteomic study on a mouse model indicated that the inflammation in hypothalamus was associated with AN and obesity (10).
Despite the presence of the conducted molecular studies, the underlying molecular mechanisms was remained elusive (11). One of the widespread used new disciplines in molecular studies is proteinprotein interaction (PPI) network analysis used for highlighting genes with further vital roles in terms of centrality properties. The role of these contributors is in the network integrity and function that may be associated with the abnormal phenotype or a disease condition (12). In fact, any small changes in these critical elements may trigger vast amount of interaction changes in the whole interacting profile (13).
Therefore, introducing a panel of such main genes may be beneficial for disease molecular basis understanding and consequently deciphering the underlying mechanisms (14). On the other hand, PPI network analysis of AN has been remained to study. In this regard, introduced AN genes by previous studies and available in String database were evaluated as a protein-protein interaction network for determination of the most central ones in the onset and development of anorexia disorder.

Material and Methods
In our study, Cytoscape 3.4.0 visualizes the protein-protein interaction network for anorexia.
In a way that, STRING DB V 10.5 (http://stringdb.org/), Plug-in was applied for interaction query for the studied disorder. This application provides three different sources for the interaction mapping including protein query, PubMed query, and disease query (15). By using of disease database here, the interaction profile of AN was retrieved.
The all known genes related to AN from the sources linked to STRING were detected as query genes.
The confidence score for interactions was set to 0.5 and other information for genes such as their relevant tissues was also provided. Furthermore, the centrality analysis was performed by the usage of Network Analyzer, well-integrated in Cytoscape (16). Two important parameters including degree and betweenness centrality are considered for the network examination by the mentioned software. In addition, for assessing functional annotation of the hub genes, ClueGO+CluePedia was applied.
In this algorithm, the linked biological processes (BPs) and molecular function (MF) for the hubs are presented as groups of terms. The terms are interconnected with the assigned kappa score. The statistical analysis used here was as follows: Kappa score = 0.5 Corrected P-value< 0.05 Number of genes per term =3 Percentage for the queried terms =4 Grouping level: Min= 2, Max= 8 Bonferroni step down was the used test for P-value correction. Moreover, two-sided (enrichment/ depletion) tests based on hypergeometric distribution for terms and groups were selected (17,18).

Results
For the network query, the number of requested  Table 1.

Interaction Network Prediction and Analysis of Anorexia Nervosa
Iran J Child Neurol. Summer 2019 Vol. 13 No. 3

Only two of six bottlenecks (BDNF and PTH) were included in the network
In this sense, the associated data for isolated bottleneck nodes in Figure 2-B are tabulated in Table 2. The enrichment analysis for the hub nodes was based on molecular functions and biological processes that were performed by ClueGO from GO Database (Figures 3 and   4).

Discussion
As anorexia is one of the complex and lethal mental conditions (19), studying molecular profile can help to better understand its underlying mechanisms (20,21). One of the new concepts that provide more information with regard to interaction pattern of molecular basis is analyzing PPI map (22). Here,  Table 2. The isolated genes are with very low rank of degree.
This supports the fact that high degree (17) genes are more essential than bottlenecks in our network.
Plasma leptin and insulin levels were decreased in the women with AN (27). Prominent role of insulin and also glucose in AN is a well-known fact emphasized by scientist in the about 30 yr ago (28).
Furthermore, regulation of behavior, regulation of carbohydrate biosynthetic process, and regulation of appetite are the foremost predicted BPs for our hub genes (Figures 3 and 4). Any alteration in the hub genes may disrupt the underlying processes in Figure 3. Similarly, the principal MFs for these hubs are extracted as neuropeptide receptor binding, neuropeptide hormone activity, and hormone activity. Thus, the prominent contribution of hormones and also behavioral alteration in AN is highlighted in this study. It seems beside feeding and metabolic management, behavioral treatment is necessary to reduce problems of patients. This finding consists of the report which introduces cognitive behavioral therapy and interpersonal psychotherapy as effective treatment methods for anorexia (29). should be applied to support this claim.