Microarray analysis of peripheral blood lymphocytes from ALS patients and the SAFE detection of the KEGG ALS pathway
1 Department of Neurology, ALS Biomarker Laboratory - James G Cannon Research Center, Carolinas Medical Center, 1542 Garden Terrace, Charlotte, NC 28203-6110, USA
2 Department of Neurology, Carolinas Neuromuscular/ALS-MDA Center, Carolinas Medical Center, 1010 Edgehill Road North, Charlotte, NC 28207-1885, USA
3 Department of Neurology, University of North Carolina School of Medicine-Charlotte Campus, Carolinas Medical Center, 1000 Blythe Blvd, Charlotte, NC 28203-5812, USA
4 Department of Biostatistics, University of North Carolina at Chapel Hill, 4115B McGavran-Greenberg Hall, CB #7420, Chapel Hill, NC 27599-7420, USA
BMC Medical Genomics 2011, 4:74 doi:10.1186/1755-8794-4-74Published: 25 October 2011
Sporadic amyotrophic lateral sclerosis (sALS) is a motor neuron disease with poorly understood etiology. Results of gene expression profiling studies of whole blood from ALS patients have not been validated and are difficult to relate to ALS pathogenesis because gene expression profiles depend on the relative abundance of the different cell types present in whole blood. We conducted microarray analyses using Agilent Human Whole Genome 4 × 44k Arrays on a more homogeneous cell population, namely purified peripheral blood lymphocytes (PBLs), from ALS patients and healthy controls to identify molecular signatures possibly relevant to ALS pathogenesis.
Differentially expressed genes were determined by LIMMA (Linear Models for MicroArray) and SAM (Significance Analysis of Microarrays) analyses. The SAFE (Significance Analysis of Function and Expression) procedure was used to identify molecular pathway perturbations. Proteasome inhibition assays were conducted on cultured peripheral blood mononuclear cells (PBMCs) from ALS patients to confirm alteration of the Ubiquitin/Proteasome System (UPS).
For the first time, using SAFE in a global gene ontology analysis (gene set size 5-100), we show significant perturbation of the KEGG (Kyoto Encyclopedia of Genes and Genomes) ALS pathway of motor neuron degeneration in PBLs from ALS patients. This was the only KEGG disease pathway significantly upregulated among 25, and contributing genes, including SOD1, represented 54% of the encoded proteins or protein complexes of the KEGG ALS pathway. Further SAFE analysis, including gene set sizes >100, showed that only neurodegenerative diseases (4 out of 34 disease pathways) including ALS were significantly upregulated. Changes in UBR2 expression correlated inversely with time since onset of disease and directly with ALSFRS-R, implying that UBR2 was increased early in the course of ALS. Cultured PBMCs from ALS patients accumulated more ubiquitinated proteins than PBMCs from healthy controls in a serum-dependent manner confirming changes in this pathway.
Our study indicates that PBLs from sALS patients are strong responders to systemic signals or local signals acquired by cell trafficking, representing changes in gene expression similar to those present in brain and spinal cord of sALS patients. PBLs may provide a useful means to study ALS pathogenesis.