Figure 3.2: Differential gene expression (DGE) results for maternal HIV. Differential gene expres- sion (DGE) results for maternal HIV. DGE was adjusted for Smoking, Maternal alcohol, Gender, Gestational age, Mode of delivery, Ethnicity and PTSD. (a) Volcano plot presents the outcome of log2 fold-change in x axis and-log10 of p-value significance in y axis for the differentially expressed genes for maternal HIV. Thin red dotted line marks p-value 0.005 and the thick orange dotted line marks p-value 0.001. (b) Top 20 genes from the results of DGE analysis for HIV exposure. Results presented as log2 fold-change (logFC), p-value, and FDR p-adjusted-value for each gene. The red dotted line indicates p-adjusted-value<0.05.
3.2.2 Enrichment Analysis of Differentially Expressed Genes for Maternal HIV
fGSEA identified enrichment of 243 GO biological processes and 24 KEGG pathways (Appendix B - Tables B.2 and B.3) differentially enriched between HEU and unexposed infants. The 20 GO biological processes (FDR adjusted p-value < 0.05) are presented in Figure 3.3 and the top 11 KEGG pathways (FDR adjusted p-value <0.05) are shown in Figure 3.4.
Pathway Gene ranks NES pval padj
GO_COTRANSLATIONAL_PROTEIN_TARGETING_TO_MEMBRANE 1.81 1.7e−04 2.1e−02
GO_REDUCTION_OF_FOOD_INTAKE_IN_RESPONSE_TO_DIETARY_EXCESS 1.47 3.9e−04 2.1e−02
GO_NUCLEOSIDE_BISPHOSPHATE_CATABOLIC_PROCESS 1.47 3.9e−04 2.1e−02
GO_PROTEIN_LOCALIZATION_TO_ENDOPLASMIC_RETICULUM 1.67 6.5e−04 2.7e−02
GO_MITOTIC_NUCLEAR_ENVELOPE_DISASSEMBLY 1.74 9.3e−04 3.5e−02
GO_ESTABLISHMENT_OF_PROTEIN_LOCALIZATION_TO_ENDOPLASMIC_RETICULUM 1.68 9.8e−04 3.7e−02
GO_VIRAL_GENE_EXPRESSION 1.62 1.1e−03 4.0e−02
GO_POSITIVE_REGULATION_OF_NECROTIC_CELL_DEATH 1.70 1.3e−03 4.4e−02
GO_NEGATIVE_REGULATION_OF_AXON_GUIDANCE 1.80 1.7e−03 5.0e−02
GO_RIBOSOMAL_SMALL_SUBUNIT_ASSEMBLY 1.85 1.7e−03 5.0e−02
GO_REGULATION_OF_VACUOLE_ORGANIZATION −2.15 2.3e−04 2.1e−02
GO_NEUROINFLAMMATORY_RESPONSE −2.09 2.3e−04 2.1e−02
GO_GLIAL_CELL_ACTIVATION −2.13 2.3e−04 2.1e−02
GO_NEGATIVE_REGULATION_OF_MACROAUTOPHAGY −2.00 2.2e−04 2.1e−02
GO_REGULATION_OF_AUTOPHAGOSOME_ASSEMBLY −2.03 2.2e−04 2.1e−02
GO_LEUKOCYTE_ACTIVATION_INVOLVED_IN_INFLAMMATORY_RESPONSE −2.40 2.2e−04 2.1e−02
GO_REGULATION_OF_MICROGLIAL_CELL_ACTIVATION −2.03 2.2e−04 2.1e−02
GO_POSITIVE_REGULATION_OF_VACUOLE_ORGANIZATION −1.97 2.2e−04 2.1e−02
GO_DENDRITIC_CELL_MIGRATION −2.06 2.2e−04 2.1e−02
GO_MYELOID_DENDRITIC_CELL_DIFFERENTIATION −1.82 2.2e−04 2.1e−02
0 2500 5000 7500 10000
Figure 3.3: Enrichment table of top 20 GO (Gene Ontology) biological processes for maternal HIV.
Enrichment table of top 20 GO (Gene Ontology) biological processes enriched by differentially expressed genes for maternal HIV exposure with their normalized enrichment scores, p-value and p-adjustment-value. The results are based on p-adjusted-value < 0.05. Genes are presented as their ranking score based on the results of DGE analysis.
3.2.3 Weighted Gene Co-Expression Network Analysis (WGCNA) for Identi- fying Highly Co-Expressed Genes
WGCNA identified 14 modules shown in Figure 3.5 using the exponential power, β = 12. The results using different β values, deep split options, and module merging options are reported in Appendix B - Figures B.1, B.2, B.3, B.4, and B.5. HEU was positively associated with the black (termed M1) and purple (M10) modules and negatively with the yellow module (M14). MEs were correlated with all clinical measures along with HIV exposure status. Consistent with the previous results, HEU was found to be most strongly correlated with M1, M10 and M14 in comparison to other exposures (Figure 3.6).
The comparisons of the modules to each other based on DEGs for HEU in terms of MS are shown in Figures 3.7(a) and B.6. Again, consistent with previous results, the M1 and M14 modules were observed to have the greatest enrichment for DEGs associated with HEU. Using a kME>0.7, 177 and 191 hub genes were identified from M1 and M14 respectively (Appendix B - Ta- ble B.4). The genes most highly associated with HEU,LOC100127993, LOC643433, LOC646766, and LOC647856 from M1 andADIPOR1, FOXO4,and HEMGN from M14 were identified using GS>0.2 and kME>0.8 (Figure B.6). Over-representation analysis on the modules identified by
Pathway Gene ranks NES pval padj
KEGG_RIBOSOME 2.14 1.7e−04 3.8e−03
KEGG_FC_GAMMA_R_MEDIATED_PHAGOCYTOSIS −2.06 2.4e−04 3.8e−03
KEGG_B_CELL_RECEPTOR_SIGNALING_PATHWAY −1.86 2.3e−04 3.8e−03
KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION −2.14 2.3e−04 3.8e−03
KEGG_CELL_ADHESION_MOLECULES_CAMS −2.05 2.3e−04 3.8e−03
KEGG_LEISHMANIA_INFECTION −2.13 2.3e−04 3.8e−03
KEGG_HEMATOPOIETIC_CELL_LINEAGE −1.94 2.3e−04 3.8e−03
KEGG_GRAFT_VERSUS_HOST_DISEASE −2.02 2.2e−04 3.8e−03
KEGG_TYPE_I_DIABETES_MELLITUS −2.00 2.2e−04 3.8e−03
KEGG_ALLOGRAFT_REJECTION −2.06 2.2e−04 3.8e−03
KEGG_AUTOIMMUNE_THYROID_DISEASE −2.06 2.2e−04 3.8e−03
0 2500 5000 7500 10000
Figure 3.4: Enrichment table of top 11 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways for maternal HIV. Enrichment table of top 11 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways enriched by differentially expressed genes for maternal HIV exposure with their normalized enrichment scores, p-value and p-adjustment-value. The results are based on p-adjusted-value <0.05. Genes are presented as their ranking score based on the results of DGE analysis. It shows the single positively enriched and top 10 negatively enriched pathways after filtering by p-adjusted-value <0.05 and ordering by p-value.
WGCNA using the DEGs for HEU showed that there was over-representation of HEU DEGs in M14 and M1 (Figure 3.7(b)).
3.2.4 Association of HEU Associated Highly Co-Expressed Genes with Lung Function
The results of LR with tidal volume at 6 weeks of age for 177 hub genes of M1 after adjustment for weight-for-age z-score, gestational age, male gender, and active smoking factors are reported in Appendix B - Table B.5. The genesLOC728428 and LOC729500 were significantly associated with increased tidal volume (2.3-3.7mL, p-value<0.05). For the 191 hub genes of M14, the results of the same model with tidal volume at 6 weeks are shown in Appendix B - Table B.6. The genes LOC730455, CTNNAL1, and ARL4Awere significant (1.8-2.3mL, p-value <0.05).
Linear regression for the hub genes of M14 with tidal volume at age of 2 years after adjusted with the confounding variables identified 42 significant (p-value<0.05) genes shown in Appendix B - Table B.7. For the hub genes of M1 no genes were significantly associated with increase in tidal volume (5.0-13.1mL, p-value<0.05) at age 2 years. Moreover, the results of functional enrichment
0.50.60.70.80.91.0
Gene Dendrogram and Module Colors (Beta=12)
hclust (*, "average") as.dist(dissTOMPre)
Height
Module colors
HIV
Figure 3.5: Association of maternal HIV with co-expressed gene clusters (i.e., modules) identified by WGCNA. Association of maternal HIV with co-expressed gene clusters (i.e., modules) identified by Weighted Gene Co-expression Network Analysis (WGCNA). WGCNA was performed on the expression of 10,705 genes of 144 samples. This figure represents a gene dendrogram (a tree representation of the co-expressed genes) at top, a module colour band at the right after the tree and a gene-phenotype (i.e., maternal HIV) colour band at the bottom. The module colour band indicates 14 different modules and the gene-phenotype colour band shows the association of maternal HIV with different modules. In gene-phenotype colour band red represents strong positive association and blue represents strong negative association with maternal HIV exposure.
analysis using the hub genes from M1 and M14 are reported in Figures 3.8(a) and 3.8)(b). These results demonstrate the relationship of some of the hub genes with the offspring lung function at a later part of their life, which is observed through these genes’ impacts on some immune system oriented REACTOME pathways.