Category: Biomarker

Exploration of the gene expression correlates of chronic unexplained fatigue using factor analysis

Abstract:

OBJECTIVE: To identify biomarkers of chronic fatigue syndrome (CFS) and related disorders through analysis of microarray data, pathology test results and self-report symptom profiles.

METHOD: To empirically derive the symptom domains of the illnesses, factor analysis was performed on responses to self-report questionnaires (multidimensional fatigue inventory, Centers for Disease Control and Prevention (CDC) symptom inventory and Zung depression scale) before validation with independent datasets. Gene expression patterns that distinguished subjects across each factor dimension were then sought.

RESULTS: A four-factor solution was favored, featuring ‘fatigue’ and ‘mood disturbance’ factors. Scores on these factors correlated with measures of disability on the Short Form (SF)-36. A total of 57 genes that distinguished subjects along each factor dimension were identified, although the separation was significant only for subjects beyond the extreme (15th and 85th) percentiles of severity. Clustering of laboratory parameters with expression of these genes revealed associations with serum measurements of pH, electrolytes, glucose, urea, creatinine, and liver enzymes (aspartate amino transferase [AST] and alanine amino transferase [AST]); as well as hematocrit and white cell count.

CONCLUSION: CFS is a complex syndrome that cannot simply be associated with changes in individual laboratory tests or expression levels of individual genes. No clear association with gene expression and individual symptom domains was found. However, analysis of such multifacetted datasets is likely to be an important means to elucidate the pathogenesis of CFS.

 

Source: Fostel J, Boneva R, Lloyd A. Exploration of the gene expression correlates of chronic unexplained fatigue using factor analysis. Pharmacogenomics. 2006 Apr;7(3):441-54. https://www.ncbi.nlm.nih.gov/pubmed/16610954

 

Challenges for molecular profiling of chronic fatigue syndrome

Abstract:

Chronic fatigue syndrome (CFS) is prevalent, disabling and costly. Despite extensive literature describing the epidemiology and clinical aspects of CFS, it has been recalcitrant to diagnostic biomarker discovery and therapeutic intervention. This is due to the fact that CFS is a complex illness defined by self-reported symptoms and diagnosed by the exclusion of medical and psychiatric diseases that may explain the symptoms.

Studies attempting to dissect the pathophysiology are challenging to design as CFS affects multiple body systems, making the choice of which system to study dependent on an investigators area of expertise. However, the peripheral blood appears to be facilitating the molecular profiling of several diseases, such as CFS, that involve bodywide perturbations that are mediated by the CNS. Successful molecular profiling of CFS will require the integration of genetic, genomic and proteomic data with environmental and behavioral data to define the heterogeneity in order to optimize intervention.

 

Source: Vernon SD, Whistler T, Aslakson E, Rajeevan M, Reeves WC. Challenges for molecular profiling of chronic fatigue syndrome. Pharmacogenomics. 2006 Mar;7(2):211-8. https://www.ncbi.nlm.nih.gov/pubmed/16515400

 

Chronic fatigue syndrome is associated with diminished intracellular perforin

Abstract:

Chronic fatigue syndrome (CFS) is an illness characterized by unexplained and prolonged fatigue that is often accompanied by abnormalities of immune, endocrine and cognitive functions. Diminished natural killer cell cytotoxicity (NKCC) is a frequently reported finding. However, the molecular basis of this defect of in vitro cytotoxicy has not been described.

Perforin is a protein found within intracellular granules of NK and cytotoxic T cells and is a key factor in the lytic processes mediated by these cells. Quantitative fluorescence flow cytometry was used to the intracellular perforin content in CFS subjects and healthy controls.

A significant reduction in the NK cell associated perforin levels in samples from CFS patients, compared to healthy controls, was observed. There was also an indication of a reduced perforin level within the cytotoxic T cells of CFS subjects, providing the first evidence, to our knowledge, to suggest a T cell associated cytotoxic deficit in CFS. Because perforin is important in immune surveillance and homeostasis of the immune system, its deficiency may prove to be an important factor in the pathogenesis of CFS and its analysis may prove useful as a biomarker in the study of CFS.

 

Source: Maher KJ, Klimas NG, Fletcher MA. Chronic fatigue syndrome is associated with diminished intracellular perforin. Clin Exp Immunol. 2005 Dec;142(3):505-11. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1440524/ (Full article)

 

37-Kilodalton/83-kilodalton RNase L isoform ratio in peripheral blood mononuclear cells: analytical performance and relevance for chronic fatigue syndrome

Comment on: RNase L levels in peripheral blood mononuclear cells: 37-kilodalton/83-kilodalton isoform ratio is a potential test for chronic fatigue syndrome.[Clin Diagn Lab Immunol. 2003]

 

A French group has reported results (5) supporting the use of the RNase L 37-kDa/83-kDa ratio (37/83 R) in peripheral blood mononuclear cells (PBMC) as a diagnostic test for chronic fatigue syndrome (CFS). More recently (6), the same group cautioned about the diagnostic value of the 37/83 R, based on a small patient follow-up study which was likely to indicate analytical variability among duplicate assays, lack of reproducibility over time, and a weak correlation with the multidimensional fatigue inventory (MFI) score. Because of our long-term experience with this assay, we would like to offer some comments.

First, we tested the analytical performance of the 37/83 R assay according to CLSI (formerly NCCLS) procedure EP5-A (4), with control samples at three different levels made of extracts of the monocytic U937 cell line spiked with various concentrations of recombinant RNase L. The guideline protocol involves assaying the samples in duplicate twice daily over a total period of 20 days. The results summarized in Table Table11 indicate that both within- and between-run variation does not exceed 13%. In another series of experiments, we assayed eight patient samples in duplicate (average 37/83 R ranging from 0.5 to 245). Although in accordance with the NCCLS protocol results, the variation did not exceed 12% for samples with 37/83 R levels up to 20, and it rose significantly to 30% and more for samples with 37/83 R levels above 20. This should be expected, because beyond this level, more than 70% of the 83-kDa isoform is cleaved, and consequently, the faint 83-kDa band is difficult to scan with accuracy. Thus, in our opinion, the lower level of correlation between the duplicate assay results observed with the CFS group versus those with the controls (6) reflects the prevalence of high 37/83 R levels in the CFS group rather than a low test reproducibility as claimed by these authors. This is further supported by the good correlation found for the control group (r = 0.95). During validation, the lowest detectable ratio measured with a sample containing the 83-kDa isoform only was estimated (3 independent experiments with 26 replicates each) to be 0.13 ± 0.06 (average ± three standard deviations). Thus, the clinical cutoff ratio of 0.4 found by the authors (5, 6) to best discriminate CFS patients from controls falls within the measurable range.

You can read the rest of this comment here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1247842/

 

Source: Frémont M, Vaeyens F, Herst CV, De Meirleir K, Englebienne P. 37-Kilodalton/83-kilodalton RNase L isoform ratio in peripheral blood mononuclear cells: analytical performance and relevance for chronic fatigue syndrome. Clin Diagn Lab Immunol. 2005 Oct;12(10):1259-60; author reply 1260. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1247842/ (Full article)

 

CFSUM1 and CFSUM2 in urine from patients with chronic fatigue syndrome are methodological artefacts

Abstract:

McGregor et al. reported increased levels of an unidentified urinary compound (CFSUM1) in patients with chronic fatigue syndrome (CFS), with reduced excretion of another unidentified compound (CFSUM2), and suggested the possibility of chemical or metabolic ‘markers’ for CFS. The identity of CFSUM1 as reported was erroneous and the identities of these compounds have remained unknown until now.

Urine samples were obtained from 30 patients with ME/CFS, 30 age- and sex-matched healthy controls, 20 control patients with depression and 22 control patients with rheumatoid arthritis. Samples were prepared using the published methods of McGregor et al. to produce heptafluorobutyryl-isobutyl derivatives of urinary metabolites. Alternative preparations utilised isopropyl, n-butyl and trifluoroacetyl derivatives. These were separated and identified using gas chromatography-mass spectrometry.

CFSUM2 was identified as being partially derivatised [isobutyl ester-mono-heptafluorobutyryl (HFB)] serine. CFSUM1 was identified as partially derivatised pyroglutamic acid, being the isobutyl ester without formation of a HFB derivative.

Both CFSUM1 and CFSUM2 are artefacts of the sample preparation procedure and previously reported quantitative abnormalities of CFSUM1 and CFSUM2 in urine from patients with ME/CFS are also artefactual. Pyroglutamic acid may be of primarily dietary origin. The methods used cannot provide reliable qualitative or quantitative data on urinary metabolites. No clinical or biochemical significance can be drawn between these compounds in ME/CFS or any other clinical conditions.

 

Source: Chalmers RA, Jones MG, Goodwin CS, Amjad S. CFSUM1 and CFSUM2 in urine from patients with chronic fatigue syndrome are methodological artefacts. Clin Chim Acta. 2006 Feb;364(1-2):148-58. Epub 2005 Aug 10. http://www.ncbi.nlm.nih.gov/pubmed/16095585

 

Urinary and plasma organic acids and amino acids in chronic fatigue syndrome

Abstract:

Previous work by others have suggested the occurrence of one or more chemical or metabolic ‘markers’ for ME/CFS including specific amino acids and organic acids and a number of unidentified compounds (CFSUM1, CFSUM2). We have shown elsewhere that CFSUM1 is partially derivatised pyroglutamic acid and CFSUM2 partially derivatised serine and have suggested and demonstrated that the analytical methods used were unsuitable to identify or to accurately quantify urinary metabolites. We have now made a detailed analysis of plasma and urinary amino acids and of urinary organic acids from patients with ME/CFS and from three control groups.

Fasting blood plasma and timed urine samples were obtained from 31 patients with CFS, 31 age and sex-matched healthy controls, 15 patients with depression and 22 patients with rheumatoid arthritis. Plasma and urinary amino acids and urinary organic acids were determined using established and validated methods and data compared by statistical analysis. None of the previously reported abnormalities in urinary amino acids or of organic acids could be confirmed.

Results however provide some evidence in patients with ME/CFS for underlying inflammatory disease and for reduced intramuscular collagen with a lowered threshold for muscle micro-injury. These factors in combination may provide a basis for the fatigue and muscle pain that are the major symptoms in these patients.

 

Source: Jones MG, Cooper E, Amjad S, Goodwin CS, Barron JL, Chalmers RA. Urinary and plasma organic acids and amino acids in chronic fatigue syndrome. Clin Chim Acta. 2005 Nov;361(1-2):150-8. http://www.ncbi.nlm.nih.gov/pubmed/15992788

 

Exercise capacity and immune function in male and female patients with chronic fatigue syndrome (CFS)

Abstract:

Hyperactivition of an unwanted cellular cascade by the immune-related protein RNase L has been linked to reduced exercise capacity in persons with chronic fatigue syndrome (CFS). This investigation compares exercise capacities of CFS patients with deregulation of the RNase L pathway and CFS patients with normal regulation, while controlling for potentially confounding gender effects.

Thirty-five male and seventy-one female CFS patients performed graded exercise tests to voluntary exhaustion. Measures of peak VO2, peak heart rate, body mass index, perceived exertion, and respiratory quotient were entered into a two-way factorial analysis with gender and immune status as independent variables. A significant multivariate main effect was found for immune status (p < 0.01), with no gender effect or interaction.

Follow-up analyses identified VO2(peak) as contributing most to the difference. These results implicate abnormal immune activity in the pathology of exercise intolerance in CFS and are consistent with a channelopathy involving oxidative stress and nitric oxide-related toxicity.

 

Source: Snell CR, Vanness JM, Strayer DR, Stevens SR. Exercise capacity and immune function in male and female patients with chronic fatigue syndrome (CFS). In Vivo. 2005 Mar-Apr;19(2):387-90. http://iv.iiarjournals.org/content/19/2/387.long (Full article)

 

Variability of the RNase L isoform ratio (37 kiloDaltons/83 kiloDaltons) in diagnosis of chronic fatigue syndrome

Chronic fatigue syndrome (CFS) is a disorder characterized by debilitating fatigue whose etiology and pathophysiology remain unclear. Previous studies showed abnormalities of the RNase L pathway in peripheral blood mononuclear cells (PBMC) from patients with CFS (1, 2). The ratio of RNase L isoforms (37 kDa/83 kDa ratio [37/83 R]) has therefore been proposed as a potential biochemical marker of CFS, with a sensitivity of 91% and a specificity of 71% when the cutoff ratio was 0.4 (3).

You can read the rest of this article here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549313/

 

Source: Tiev KP, Briant M, Ziani M, Cabane J, Demettre E, Lebleu B. Variability of the RNase L isoform ratio (37 kiloDaltons/83 kiloDaltons) in diagnosis of chronic fatigue syndrome. Clin Diagn Lab Immunol. 2005 Feb;12(2):366. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549313/ (Full article)

 

Urinary electrophoretic profiles from chronic fatigue syndrome and chronic fatigue syndrome/fibromyalgia patients: a pilot study for achieving their normalization

Abstract:

Aim of our study was to determine if there were distinct, disease-related patterns of urinary analytes in chronic fatigue syndrome (CFS) and chronic fatigue syndrome/fibromyalgia (CFS/FM) compared to normal controls (NC).

Urine was collected from these subjects for two consecutive 24 h periods and aliquots were submitted to micellar electrokinetic chromatography (MEKC). To compensate for the differences in peak migration times, these were normalized from the 35 min duration of run to a 100-point scale, and each peak was assigned its normalized time measure. Peak heights were also normalized by dividing the mAU by that of the internal standard (creatinine) and multiplying by 100. MEKC with normalization for peak height and migration time generated comparable results within each of the patient groups.

CFS/FM and CFS had significant differences in peaks compared to NC that may be of significance as biomarkers of illnesses.

 

Source: Casado B, Zanone C, Annovazzi L, Iadarola P, Whalen G, Baraniuk JN. Urinary electrophoretic profiles from chronic fatigue syndrome and chronic fatigue syndrome/fibromyalgia patients: a pilot study for achieving their normalization. J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Jan 5;814(1):43-51. http://www.ncbi.nlm.nih.gov/pubmed/15607706

 

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

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 nonredundant 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 downregulated, and the remaining five genes (cathepsin B, DNA polymerase epsilon4, novel EST PBMC191MSt, heparanase precursor, and ORF2/L1 element) were upregulated 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.

 

Source: Steinau M, Unger ER, Vernon SD, Jones JF, Rajeevan MS. Differential-display PCR of peripheral blood for biomarker discovery in chronic fatigue syndrome. J Mol Med (Berl). 2004 Nov;82(11):750-5. Epub 2004 Oct 14. http://www.ncbi.nlm.nih.gov/pubmed/15490094