Variations in the progranulin gene affect global gene expression in frontotemporal lobar degeneration

Abstract

Frontotemporal lobar degeneration is a fatal neurodegenerative disease that results in progressive decline in behavior, executive function and sometimes language. Disease mechanisms remain poorly understood. Recently, however, the DNA- and RNA-binding protein TDP-43 has been identified as the major protein present in the hallmark inclusion bodies of frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U), suggesting a role for transcriptional dysregulation in FTLD-U pathophysiology. Using the Affymetrix U133A microarray platform, we profiled global gene expression in both histopathologically affected and unaffected areas of human FTLD-U brains. We then characterized differential gene expression with biological pathway analyses, cluster and principal component analyses, and subgroup analyses based on brain region and progranulin (GRN) gene status. Comparing 17 FTLD-U brains to 11 controls, we identified 414 upregulated and 210 downregulated genes in frontal cortex (P-value < 0.001). Moreover, cluster and principal component analyses revealed that samples with mutations or possibly pathogenic variations in the GRN gene (GRN+, 7/17) had an expression signature that was distinct from both normal controls and FTLD-U samples lacking GRN gene variations (GRN-, 10/17). Within the subgroup of GRN+ FTLD-U, we found >1300 dysregulated genes in frontal cortex (P-value < 0.001), many participating in pathways uniquely dysregulated in the GRN+ cases. Our findings demonstrate a distinct molecular phenotype for GRN+ FTLD-U, not readily apparent on clinical or histopathological examination, suggesting distinct pathophysiological mechanisms for GRN+ and GRN- subtypes of FTLD-U. In addition, these data from a large number of human brains provide a valuable resource for future testing of disease hypotheses.

Figure 1.

Cluster analysis of all samples shows a distinct expression signature for cerebellum. Hierarchical dendrogram produced by clustering of expression levels for 56 brain samples (columns) and 22 277 transcripts (rows), using Spearman correlation coefficients. With one exception, all 17 cerebellar samples (red) regardless of disease status fell into one branch (bold), while samples from frontal cortex (blue) and hippocampus (yellow) were admixed. Disease status for each sample is shown as well, with orange denoting normal cases, purple denoting FTLD-U cases without progranulin gene abnormalities (GRN− FTLD-U) and teal denoting FTLD-U cases with progranulin gene abnormalities (GRN+ FTLD-U). Heatmap tiles show standardized expression levels of individual genes with red denoting high expression, grey denoting medium expression and blue denoting low expression levels.

Figure 2.

Number of dysregulated genes identified in human cortex microarray studies of various neurodegenerative diseases. Data from present study (FTLD-U) was re-analyzed using the same statistical conditions as each comparison study (–31). See Supplementary Material, Table S3 for details of other studies. The black portion of each bar represents the number of upregulated genes in disease compared to control, while the white portion represents the number of downregulated genes in disease compared to control. Many more gene changes were found in the present FTLD-U study than in studies of AD or ALS.

Figure 3.

Cluster analysis and principal components analysis of samples from histopathologically affected brain regions show a distinct expression signature for GRN+ FTLD-U. (A) Hierarchical dendrogram produced by clustering of expression levels for 39 brain samples from frontal cortex or hippocampus (columns) and 22 277 transcripts (rows), using Spearman correlation coefficients. With two exceptions, all 11 GRN+ FTLD-U samples (yellow) fell into one branch (bold), while samples from GRN− FLTD-U (red) and normal controls (blue) were admixed. Brain region for each sample is shown as well, with orange denoting frontal cortex samples and teal denoting hippocampal samples. Heatmap tiles show standardized expression levels of individual genes with red denoting high expression, grey denoting medium expression and blue denoting low expression levels. (B) Principal components analysis of frontal and hippocampal samples using 22 277 transcripts also revealed a distinct expression signature for GRN+ FTLD-U. GRN+ FTLD-U samples (yellow) were characterized by a low score along the first principal component (X-axis) and a high score along the second principal component (Y-axis), separating them from GRN− FTLD-U samples (red) and normal samples (blue). Arrows indicate the two samples that were more distant in global gene expression by cluster analysis. Within GRN+ FTLD-U cases, samples bearing truncation mutations (squares) and samples bearing variants of unknown significance (triangles) were admixed. R493X=predicted protein change for indicated truncation mutant. R418X=predicted protein change for indicated truncation mutant.

Figure 4.

Validation of microarray results by quantitative reverse transcription real-time PCR (QRT–PCR). Relative expression levels of genes are indicated by the fold changes in expression level for FTLD-U samples with progranulin gene abnormalities (GRN+), and FTLD-U samples without progranulin gene abnormalities (GRN−). For QRT–PCR, average fold changes and standard deviations of three cases in each group are shown. PCR reactions were performed in duplicate. GLRB, glycine receptor beta subunit; GRM5, metabotropic glutamate receptor 5; HDAC1, histone deacetylase 1; HSPA2, heat shock protein 2 (70 kD); HTR2A, serotonin receptor 2A; ID4, inhibitor of DNA binding 4; NOTCH2, Notch homolog (Drosophila) 2.