This tutorial describes a simple example showing how eDGAR collects different types of relations among the genes involved in a disease associated to multiple genes. Hypoparathyroidism is an endocrine deficiency disease characterized by low serum calcium levels, elevated serum phosphorus levels and absent or low levels of parathyroid hormone (PTH) in blood. The metabolism of the patient may be altered: the vitamin D supply is inadequate and the magnesium metabolism is irregular. In some clinical panel, hypocalcemia can lead to dramatic effects such as tetany, seizures, altered mental status, refractory congestive heart failure, or stridor.

The user may search for hypoparathyroidism in two different ways:
1) Browsing the main table: the OMIM ID (146200) or the disease name (hypoparathyroidism) can be used to narrow the search.
2) From the search page, entering the OMIM ID (146200), as shown in Figure 1.

Tutorial 1
Fig1. The Search page.

In both cases, the user is redirect to the Disease page relative to hypoparathyroidism.

In the first table, called "Gene-disease associations table" (Figure 2), eDGAR reports all the genes associated to the disease; in this case three different genes: GMC2, PTH and CASR. The second column of the table highlights that GCM2 and PTH are both reported in OMIM, ClinVar and Humsavar while and CASR is reported only in ClinVar.

Tutorial 2
Fig2. The gene-disease associations table.

The gene names are active links that redirect the to the corresponding eDGAR gene pages. CASR is an extracellular calcium-sensing receptor whose activity is mediated by G-proteins, PTH is the parathyroid hormone, whose function is to increase calcium level both by promoting the solution of bone salts and by preventing their renal excretion, and GCM2 (Glial cell missing homolog 2) is a probable transcriptional regulator, as to the SwissProt annotation.
The Disease page collects all the possible relations among the three genes. Black titles correspond to annotation with available information. Grey titles mark missing annotations. In the case of hypoparathyroidism, the first available feature is "The Transcription Factor (TF) annotation from TRRUST" (Figure 3). Clicking on the title, two table appear: the list of T/target gene pairs in the set and the list of genes coregulated by the same TF. The tables report that GMC2 is a TF that regulates the expression of both PTH and CASR.

Tutorial 3
Fig3. The Transcription Factors annotation.

The "Interactions from STRING" table (Figure 4) reports that PTH and CASR are in direct interaction, labelled as "binding" and "expression". Under the table a link visualizes the graph of the interactions. Two types of graphs are available:
1) the graph of the direct interactions (Figure 5),
2) the graph collecting both direct and indirect interactions involving genes not included in the set.

Tutorial 4
Fig4. The String interactions table.

Blue nodes correspond to genes associated to the disease while pale blue nodes correspond to genes not associated to the disease. Direct interactions are highlighted in green and indirect interactions in thinner black lines. Each node has a label, and clicking on a node you will be redirected to the gene page.

Tutorial 5
Fig5. The network of direct interactions .

When looking at the KEGG pathways annotation (Figure 6), no shared KEGG pathways are found, while NET-GE allows retrieving enriched pathways, that are visualizes in a table (sorted according to IC, by default). The most informative are "Circadian entrainment (hsa04713)", "Inflammatory mediator regulation of TRP channels (hsa04750)", "Gap junction (hsa04540)" and "Insulin secretion (hsa04911)". None of the three genes is directly involved in the four pathways; PTH and CASR are part of the networks defined by NET-GE.
Interestingly, these new annotations highlight previously reported impairments of both circadian rhythms impairment and insulin secretion associated to hypoparathyroidism.

Tutorial 6
Fig6. The NET-GE enrichment table for KEGG annotation.

The REACTOME pathways are reported (shared terms and enriched terms by NET-GET). PTH and CASR are involved in the same REACTOME pathways related to GPCR ligand binding and signaling. Two new pathways are enriched with NET-GE: Diseases of metabolism (R-HSA-5668914) and Class B/2 (Secretin family receptors) (R-HSA-373080).

Similar tables are available for the three sub-ontologyes of GO: molecular function, biological process and cellular component.

In particular, for the biological process, the shared GO terms with the highest IC values are "response to vitamin D" and "response to fibroblast growth factor", both involving CASR and PTH. The response to vitamin D, whose metabolism is often altered in hypoparathyroidism, and a strict interplay between fibroblast growth factors and parathyroid hormone have been previously reported. PTH and CASR are also involved in the same REACTOME pathways related to GPCR ligand binding and signaling.
NET-GE enrichment for BP for the three genes include new terms endowed with high IC values, like "regulation of amino acid transport" and "negative regulation of muscle contraction". Some of these new annotations are related to the severe symptoms of hypothyroidisms, namely tetany and seizure.