2. Differentially Expressed Genes in Pre-Symptomatic and Symptomatic HD Patients

The top 150 over and top 150 under expressed genes, therefore a total of 300 genes were identified respectively for each group of HD patients. The DEGs for the pre-symptomatic and symptomatic HD patients, in comparison to controls, are shown in Table S1 and Table S2 respectively. DEGs that are highlighted through our pipeline and have also been already associated with HD based on the bibliography, are highlighted in Table S3. Specifically, we annotate their role in pathophysiological mechanisms such as neuro-inflammation, apoptosis, anti-oxidants and Ca²⁺ dysregulation that are involved in HD.

3. Gene Co-Expression Networks of Pre-Symptomatic and Symptomatic HD Patients

The gene co-expression networks containing the DEGs for controls versus pre-symptomatic and versus symptomatic HD patients are illustrated in (Figure 1). Blue nodes represent the genes present in pre-symptomatic HD patients, orange nodes represent genes present in symptomatic HD patients and green nodes represent genes found in both the pre-symptomatic and symptomatic HD patients. Similarly, blue, orange and green edge colour represent co-expressions observed in pre-symptomatic, symptomatic and both HD stages respectively. Additionally, network topology analysis revealed a more tightly connected co-expression network for the pre-symptomatic HD patients compared to the symptomatic, as shown by the differences in the degree, betweenness, coreness and closeness distributions in Figure 1b–e.

Figure 1. Network topological analysis of the gene co-expression. (a) Gene co-expression networks for controls versus pre-symptomatic and controls versus symptomatic HD. Blue nodes represent: the genes involved in pre-symptomatic HD, orange nodes represent: the genes involved in the symptomatic HD stage and green nodes represent the genes which appear in both HD networks. Edge colour represents co-expression in the respective groups (either or both HD stages) while edge thickness represents co-occurrence score (b–e) Distribution of the calculated centralities for the pre-symptomatic and symptomatic HD networks, i.e., (b) Degree (c) Betweenness (d) Coreness and (e) Closeness.

4. Network Rewiring between Gene Co-Expression Networks of Pre-Symptomatic and Symptomatic HD Patients Using DyNet

The gene co-expression networks for the pre-symptomatic and symptomatic HD networks were used to identify the most re-wired nodes between the pre-symptomatic and symptomatic HD networks using the DyNet Cytoscape plug-in [11]. The central reference network is an overlapping visualization view consisting of the pre-symptomatic and symptomatic HD networks as illustrated in (Figure 2). The most highly re-wired node identified based on the D_n (DyNet re-wiring) score as seen in (Figure 2) and drawn as a square node, was the calcium voltage-gated channel subunit alpha 1 I (CACNA1I). In our data we identified the CACANA1I gene to be significant with a p-value of 1.96E-05 and logFC 2.744 in pre-symptomatic HD patients. The CACNA1I gene encodes the protein CACNA1I, which is a member of a sub-family of Ca²⁺ channels. The following voltage-gated Ca²⁺ channel is involved in Ca²⁺ signalling in neurons [https://www.gehttps://www.genecards.org/], [12], anecards.org/], ^[12]Supplementary Table S4 shows the presence or absence of genes indicated by true or false in the pre-symptomatic and symptomatic networks.

Figure 2. Central reference network of the pre-symptomatic and symptomatic HD network using the Cytoscape plug-in DyNet. Dark red nodes: Most highly re-wired nodes, Medium red: Highly re-wired nodes, Light red: Least most re-wired nodes and White nodes: No re-wiring. The square node indicates the CACNA1I gene, which was the most highly re-wired node based on the DyNet re-wiring score.

A Venn diagram [https://bioinfogp.cnb.csic.es/tools/venny/], was used to identify, the DEGs and rewired genes common between the pre-symptomatic and symptomatic HD stages. The Venn Figure 3, identified, the AF198444 gene to be common between the rewired genes and DEGs symptomatic HD. Ten common genes such as CNTN6, DEK, LTN1, MST4, ZFYVE16, CEP135, DCAKD, MAP4K3, NUPL1 and RBM15 were identified between the DEGs for pre-symptomatic and DEGs for symptomatic HD. Some of the biological functions identified for the above-mentioned genes, include ubiquitin protein ligase activity, ATP binding, adenyl ribonucleotide, protein serine/threonine kinase activity, purine ribonucleotide triphosphate binding, ubiquitin-protein transferase activity, protein kinase activity and RNA binding (Table S5). Additionally, twenty-two common genes such as CACNA1I, DNAJB14, EPS8L3, HSDL2, SNRPD3, SOX12, ACLY, ATF2, BAG5, ERBB4, FOCAD, GRAMD1C, LIN7C, MIR22, MTHFR, NABP1, NRG2, OTC, PRAMEF12, SLC30A10, STAG2 and Y16709 were identified between the rewired and DEGs of pre-symptomatic. Some of the biological pathways identified include, phosphatidylinositol-4,5-bisphosphate 3-kinase activity, phosphatidylinositol bisphosphate kinase activity, cyclic adenosine monophosphate (cAMP) response element binding protein binding, protein tyrosine kinase activity, voltage-gated ion channel activity involved in regulation of postsynaptic membrane potential, voltage-gated calcium channel activity, manganese ion transmembrane transporter activity and numerous additional pathways in Table S6. Furthermore, from our studied we identified the genes of SP3 and PCNP from the DEGs symptomatic HD and CAPZA1 from the DEGs pre-symptomatic HD. The following three genes, were previous identified by [13] as biomarkers for HD.

Figure 3. Venn diagram of rewired genes and DEGs of pre-symptomatic and symptomatic HD. Venn diagram illustrates the number of common genes between the rewired genes and DEGs of the two HD stages.

The DEGs and re-wired genes obtained from DyNet were separately used as input for enrichment analysis in the EnrichR web-tool [14] for pre-symptomatic and symptomatic HD cases as well as the rewired network, to identify the common enriched pathways and genes among the co-expression networks and the re-wired HD network. The common pathways between the pre-symptomatic, symptomatic HD networks and the re-wired network were selected based on the top ranking score.

Some of the common pathways identified between the pre-symptomatic and the re-wired network include: glycosaminoglycan degradation, citrate acid cycle, ErbB signaling pathway, adherens junctions, Ca²⁺ signalling and arginine biosynthesis. Common pathways identified between the symptomatic and the rewired network include the endocytosis and proteasome pathways.

5. PathwayConnector Clustering of Pathways Identifed for Pre-Symptomatic and Symptomatic HD Patients

We further identified the HD- related pathways for the pre-symptomatic and symptomatic HD stages using PathwayConnector ^[15][15]. The pathways for each stage are shown in Table S7 and Table S8 respectively.

After enrichment analysis, mapping onto the Kyoto Encyclopedia of Genes and Genomes (KEEG) reference network and the construction of the complementary pathway-to-pathway networks for the pre-symptomatic and symptomatic HD stages, clustering was then implemented to group the final set of HD-related pathways into clusters.

6 clusters were recognized for the pre-symptomatic HD stage and 3 clusters for the symptomatic HD stage, clustering of pathways is based on the network property of edge-betweenness which defined as the number of shortest paths which go through an edge in a network [16]. The clusters are indicated by the different coloured circles and backgrounds for the pre-symptomatic and symptomatic stages respectively Figure 4.