Whole-transcriptome brain expression and exon-usage profiling in major depression and suicide: evidence for altered glial, endothelial and ATPase activity

Abstract

Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in the dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden death medication-free individuals post mortem. Using small RNA-seq, we also examined miRNA expression (nine samples per group). DeSeq2 identified 35 genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted P<0.1). In depression, altered genes include humanin-like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted P<0.1). Hypothesis-driven GO analysis suggests lower expression of genes involved in oligodendrocyte differentiation, regulation of glutamatergic neurotransmission, and oxytocin receptor expression in both suicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted P<0.1) in depression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders.

Figure 1

Top four differentially expressed genes among in MDD with and without suicide (Table 2, p<0.1 FDR corrected). Top left panel: Humanin-like 8 (MTRNR2L8) is higher in depression (MDD+MDD-S vs. CON, log2FC = 0.67, adjusted p=8.2E-5) and higher in suicide (MDD-S vs. CON+MDD, log2FC = 0.72, adjusted p=4.1E-9). Top right panel: Serpin peptidase inhibitor, clade H (heat shock protein 47), member 1 (SERPINH1) is lower in depression (log2FC=-0.72, adjusted p=2.5E-5) and lower in suicide (log2FC=-0.40). Lower left panel: Interleukin 8 (IL8) is lower in depression (log2FC=-0.53, adjusted p=0.0006). Lower right panel: Chemokine (C-C motif) ligand 4 (CCL4) is lower in depression (log2FC=-0.59, adjusted p=0.0003).

Figure 2. Details for GO gene set “chemokine receptor activity”

Each row corresponds to a gene that is included in the same set. “Element” column lists ensemble ID, “score” refers to the uncorrected p-value for the likelihood ratio test (LRT) when comparing the full model with condition (three levels: CON, MDD-S and MDD) vs. the reduced model (~ age + sex + condition vs. ~ age + sex, see methods), “QQ Score” graphs the scores: blue lines represent the observed scores, light grey line shows the expected distribution based on chance, “multifunctionality” indicates the multifunctionality of the gene. The number in parentheses is the number of annotations (e.g., GO terms) the gene has, which is roughly proportional to the multifunctionality, but the exact multifunctionality score takes into account the size of the groups to which the gene belongs, “QQ Multifunct” is similar to the QQ Score column but for the multifunctionality. If the gene set has “typical” multifunctionality, the pink line will tend to be near the grey line.

Figure 3. Details for GO gene set “cellular response to lipopolysaccharides”

See Figure 2 legend for description of columns.

Figure 4. Details for GO gene set “positive regulation of angiogenesis”

See Figure 2 legend for description of columns.

Figure 5. Exon-level expression in ATP9B after adjusting for group differences in gene-level expression

Top plot shows mean normalized counts for each of 50 splicing events (i.e. bins, or “exons”, see methods) in the ATP9B gene across for the three groups CON, MDD and MDD-S. There was a significant group effect in mean normalized counts in exon 21 (circled, adjusted p=0.087, see Table 1) after removing overall effect of gene expression and correcting for multiple comparisons across the whole exome. Blue lines indicate differentially expression exons surviving a more lenient threshold (adjusted p<0.1 correcting for only the 50 tests within this gene). The bottom row depicts 50 of 93 total unique splicing events (here referred to as “exon”) along the ATP9B gene. Exon bin 21 in reference to the 26 known ATP9B transcripts (isoforms) according to Human genome assembly GRCh37 is shown in Supplementary Figure 2.