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Chronic Fatigue Syndrome > Publications > Molecular Epidemology Publications >

Differential display PCR of peripheral blood for biomarker discovery in chronic fatigue syndrome.

Steinau M, Unger E, Vernon S, Jones J, Rajeevan M
Differential display PCR of peripheral blood for biomarker discovery in chronic fatigue syndrome
Journal of Molecular Medicine 2004;82:750-755.

Summary

As yet no definitive biomarkers for CFS have been identified. The CDC CFS Molecular Epidemiology effort is using microarray gene expression profiling as a tool to search for biomarkers. We have shown that microarray gene expression profiling shows differences between CFS cases and controls and provides evidence for sub-classification of CFS. However, microarray methods address only those genes that are known and included in the test. Another molecular method called differential display PCR has to potential to detect previously uncharacterized genes and to identify novel pathogens. This article discusses our initial experiments with differential display PCR. We found that 10 genes involved in immune regulation were differentially expressed in a patient with CFS and a control following exercise.

Abstract

We used differential-display PCR of peripheral blood mononuclear cells (PBMCs) to search for candidate biomarkers for chronic fatigue syndrome (CFS). PBMCs were collected from a subject with CFS and an age and sex-matched control before and 24 h after exercise. RNA expression profiles were generated using 46 primer combinations, and the similarity between the individuals was striking. Differentially expressed bands were excised, reamplified, and sequenced, yielding 95 non redundant sequences, of which 50 matched to known gene transcripts, 38 matched to genes with unknown functions, and 7 had no similarity to any database entry. Most (86%) of the differences between the two subjects were present at baseline. Differential expression of ten genes was verified by real-time reverse-transcription PCR: five (cystatin F, MHC class II, platelet factor 4, fetal brain expressed sequence tag, and perforin) were down regulated, and the remaining five genes (cathepsin B, DNA polymerase _4, novel EST PBMC 191 MSt, heparanase precursor, and ORF2/L1 element) were up regulated in the subject with CFS. Many of these genes have known functions in defense and immunity, thus supporting prior suggestions of immune dysregulation in the pathogenesis of CFS. Differential-display PCR is a powerful tool for identification of candidate biomarkers. Investigation of these markers in samples from well-designed epidemiological studies of CFS will be required to determine the validity of these candidate biomarkers. The real-time reverse-transcription PCR assays that we developed for assay of these biomarkers will facilitate high-throughput testing of these additional samples.

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