No abstract

Chronic fatigue syndrome (CFS) is a debilitating illness lacking consistent anatomic lesions and eluding conventional laboratory diagnosis. Demonstration of the utility of the blood for gene expression profiling and biomarker discovery would have implications into the pathophysiology of CFS. The objective of this study was to determine if gene expression profiles of peripheral blood mononuclear cells (PMBCs) could distinguish between subjects with CFS and healthy controls. Total RNA from PBMCs of five CFS cases and seventeen controls was labeled and hybridized to 1764 genes on filter arrays. Gene intensity values were analyzed by various classification algorithms and nonparametric statistical methods. The classification algorithms grouped the majority of the CFS cases together, and distinguished them from the healthy controls. Eight genes were differentially expressed in both an age-matched case-control analysis and when comparing all CFS cases to all controls. Several of the diffrentially expressed genes are associated with immunologic functions (e.g., CMRF35 antigen, IL-8, HD protein) and implicate immune dysfunction in the pathophysiology of CFS. These results successfully demonstrate the utility of the blood for gene expression profiling to distinguish subjects with CFS from healthy controls and for identifying genes that could serve as CFS biomarkers.

Chronic fatigue syndrome (CFS) is a significant public health problem of unknown etiology, the pathophysiology has not been elucidated, and there are no characteristic physical signs or laboratory abnormalities. Some studies have indicated an association of CFS with deregulation of immune functions and hypothalamicpituitary-adrenal (HPA) axis activity. In this study, we examined the association of sequence variations in the glucocorticoid receptor gene (NR3C1) with CFS because NR3C1 is a major effector of the HPA axis. There were 137 study participants (40 with CFS, 55 with insufficient symptoms or fatigue, termed as ISF, and 42 nonfatigued controls) who were clinically evaluated and identified from the general population of Wichita, KS. Nine single nucleotide polymorphisms (SNPs) in NR3C1 were tested for association of polymorphisms and haplotypes with CFS. We observed an association of multiple SNPs with chronic fatigue compared to non-fatigued (NF) subjects (P < 0.05) and found similar associations with quantitative assessments of functional impairment (by the SF-36), with fatigue (by the Multidimensional Fatigue Inventory) and with symptoms (assessed by the Centers for Disease Control Symptom Inventory). Subjects homozygous for the major allele of all associated SNPs were at increased risk for CFS with odds ratios ranging from 2.61 (CI 1.05-6.45) to 3.00 (CI 1.12-8.05). Five SNPs, covering a region of approximately 80 kb, demonstrated high linkage disequilibrium (LD) in CFS, but LD gradually declined in ISF to NF subjects. Furthermore, haplotype analysis of the region in LD identified two associated haplotypes with opposite alleles: one protective and the other conferring risk of CFS. These results demonstrate NR3C1 as a potential mediator of chronic fatigue, and implicate variations in the 5' region of NR3C1 as a possible mechanism through which the alterations in HPA axis regulation and behavioural characteristics of CFS may manifest.

Before gene expression profiling with microarray technology can be transferred to the diagnostic setting, we must have alternative approaches for synthesizing probe from limited RNA samples, and we must understand the limits of reproducibility in interpreting gene expression results. The current gold standard of probes for use with both microarrays and high-density filter arrays are synthesized from 1 g of purified poly(A)؉ RNA. We evaluated two approaches for synthesizing cDNA probes from total RNA with subsequent hybridization to high-density filter arrays: 1) reverse transcription (RT) of 5 g total RNA and 2) RT-polymerase chain reaction (RT-PCR) of 1 g total RNA, using the SMART system. The reproducibility of these two approaches was compared to the current gold standard. All three methods were highly reproducible. Triplicate experiments resulted in the following concordance correlation coefficients to evaluate reproducibility: 0.88 for the gold standard, 0.86 for cDNA probe synthesized by RT from total RNA, and 0.96 for the SMART cDNA probe synthesized from total RNA. We also compared the expression profile of 588 genes for the total RNA methods to that obtained with the gold standard. Of 150 positive genes detected by the gold standard, 97 (65%) were detected by cDNA probe synthesized by RT of total RNA, and 122 (81%) were detected by the SMART cDNA probe. We conclude that SMART cDNA probe produces highly reproducible results and yields gene expression profiles that represent the majority of transcripts detected with the gold standard. (J Mol Diag 2000, 2:124 -127)