RNase L Levels in Peripheral Blood Mononuclear Cells: 37-Kilodalton/83-Kilodalton Isoform Ratio Is a Potential Test for Chronic Fatigue Syndrome

Chronic fatigue syndrome (CFS) is a disorder characterized bydebilitating fatigue associated with immunological abnormalities.The etiology remains unclear. A low-molecular-mass (37 kDa)isoform of RNase L has been described in peripheral blood mononuclearcell (PBMC) extracts, and the ratio of two isoforms of RNaseL (37 kDa/83 kDa) has been proposed as a potential biochemicalmarker of CFS. In a prospective case-control study, we testedwhether the RNase L 37-kDa/83-kDa ratio could discriminate aSFC population. We compared the ratio of RNase L isoforms inPBMCs from 11 patients with CFS (6 women and 5 men; mean age± standard deviation, 43.2 ± 13.8 years) and PBMCsfrom 14 healthy well-matched volunteers (10 women and 4 men;age, 39.1 ± 11.6 years). A ratio of RNase L of 0.4 usedas a threshold allowed diagnosis of CFS with high sensitivity(91%; 95% confidence interval [CI], 57 to 99%) and specificity(71%; 95% CI, 41 to 90%). The positive and negative prognosticvalues were 71% (95% CI, 41 to 90%) and 91% (95% CI, 57 to 99%),respectively. In the absence of acute infection or chronic inflammation,a high RNase L ratio could distinguish CFS patients from healthyvolunteers. Additional large studies and follow-up studies arerequired to confirm the stability of this high ratio of RNaseL isoforms in a CFS group.

INTRODUCTION

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Introduction

Chronic fatigue syndrome (CFS) is an illness characterized bylong-lasting fatigue and other symptoms. Both psychologicaland biological etiological factors have been proposed, but thedisorder remains of uncertain origin. No organism could be directlyimplied in the pathogenesis of CFS, and the physiopathologyis still unclear. Because of the heterogeneous and nonspecificsymptoms of CFS, a diagnostic test is needed.

Recently, CFS has been hypothesized to result from immune dysregulation.Particularly an up-regulation of key components of the 2',5'-oligoadenylate(2-5A)-RNase L pathway has been identified in extracts of peripheralblood mononuclear cells (PBMCs) from individuals with CFS (5,6), including the presence of a low-molecular-mass form (37kDa) of RNase L, whose origin remains undetermined. Two groupssuggested that CFS occurs in an immune dysregulation backgroundthat would find its expression into a high ratio of RNase Lisoforms, and one group did not.

In a prospective case-control study, we tested whether the 37-kDa/83-kDaratio of RNase L isoforms could discriminate a SFC population.

MATERIALS AND METHODS

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Materials and Methods

Patient characteristics. The patient group consisted of 11 patients (6 women and 5 men;mean ± standard deviation [SD] age,: 43.2 ± 13.8years) fulfilling CFS criteria (2). The mean (±SD) durationof illness was 7.6 ± 6.6 years. The control group consistedof 14 matched healthy volunteers (10 women and 4 men; mean ±SD age, 39.1 ± 11.6 years). A multidimensional fatigueinventory (MFI) scale (4) was used to estimate fatigue in bothgroups. Before the inclusion, biological tests were performedfor both groups, which allowed exclusion of organic diseases.Moreover, neither infectious history nor psychiatric illnesswas noted in the month prior to the start of the study. In thetwo groups, the mean ages were comparable, and the MFI score(maximal fatigue score = 100) was significantly higher in thepatients (53.3 ± 9.4) than in healthy volunteers (6.1± 7). The clinical features of the patients are summarizedin Table 1. This study was approved by the local ethics committeeof the Hôpital Saint Antoine, Paris, France.

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TABLE 1. Clinical features of patients in this study^a

PBMC preparation. Venous blood samples were drawn in heparinized tubes. The sampleswere stored, and PBMCs were isolated within 4 h by density-gradientcentrifugation. Blood (diluted 1/1 in phosphate-buffered saline)was layered onto 1 volume of Ficoll (density, 1.080; Gibco BRL,Paisley, Scotland) and centrifuged at 500 x g for 30 min at20°C. PBMCs at the interface were collected and washed with5 volumes of phosphate-buffered saline (500 x g, 15 min, 20°C).PBMC pellets were resuspended in 5 ml of erythrocyte-lysingbuffer (155 mM NH₄Cl, 10 mM NaHCO₃ [pH 7.4], 0.1 mM EDTA), centrifuged(500 x g, 10 min, 20°C), and frozen at -80°C until use.

Radiocovalent affinity labeling and analysis of 2-5A binding proteins. A sensitive assay allowing the specific identification of 2-5Abinding proteins in biological samples without a fractionatingstep was used. PBMC extracts were prepared in the presence ofprotease inhibitors to avoid artifacts from differential proteolysis.

A 3'-oxidized 2-5A [³²P]pCp (3,000 Ci/mmol, 20,000 cpm) probewas incubated with PBMC extracts (200 µg of protein) for20 min at 4°C and for a further 20 min at room temperaturewith 20 mM sodium cyanoborohydride, allowing the covalent linkageof the probe to all 2-5A binding protein species. The extractwas then fractionated by polyacrylamide gel electrophoresis(PAGE) with sodium dodecyl sulfate (SDS) as a denaturing agent.The size distribution of 2-5A-labeled polypeptides was visualizedby autoradiography, and the relative abundance of the variousbands was calculated by densitometric analysis with the NIHImage software. NIH Image is a public domain image processingand analysis program developed at the Research Services Branchof the National Institute of Mental Health.

Statistical analysis. Sensitivity, specificity, and positive and negative prognosticvalues were calculated with different threshold ratios of RNaseL isoforms as a positive diagnosis, particularly ratios of 0.5,0.4, and 0.3. Confidence intervals (95% CIs) were estimated.The ages, durations of fatigue, numbers of CFS criteria fulfilled,and MFI scores of the groups were compared by Mann-Whitney Utest with significance at P < 0.05.

RESULTS

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Results

SDS-PAGE fractionation of 2-5A-labeled polypeptides in PBMCextracts from healthy individuals gives rise to a major 83-kDapolypeptide band. An additional low-molecular-mass (37 kDa)2-5A-labeled polypeptide can often be detected in extracts ofCFS PBMCs, in keeping with previous reports (1).

The ratio of RNase L isoforms (37 kDa/83 kDa) of 0.4 used asa cutoff allowed discrimination of CFS patients from controlswith high sensitivity (91%; 95% CI, 57 to 99%) and specificity(71%; 95% CI, 41 to 90%). The positive and negative prognosticvalues were 71% (95% CI, 57 to 99%) and 91% (95% CI, 41 to 90%),respectively. The results are summarized in Table 2. The sensitivityand specificity were lower when the other threshold values wereused.

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TABLE 2. Diagnosis of CSF with an RNase L isoform ratio of 0.4 used as the cutoff

DISCUSSION

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Discussion

Dysfunction of the 2-5A/RNase L antiviral pathway in patientswith CFS has been reported by several groups (5, 6). In particular,a 37-kDa 2-5A binding polypeptide has been identified in 88%of CFS patients compared with only 28% of healthy control subjects(1).

The present study basically agrees with these data. These datawere challenged by Gow et al. (3), who suggested that the highactivity of the RNase L found in CFS patients was just a coincidenceand probably reflected an ongoing viral infection. Indeed, reversetranscription-PCR analysis showed that RNase L mRNA levels didnot differ between a CFS group and a healthy volunteer group.However, these data are not contradictory, since Gow et al.did not take into consideration the fact that the accumulationof 37-kDa isoforms of RNase L, as described here and as reportedby De Merleir et al. (1), may result from proteolysis of RNaseL independently of RNase L mRNA level. In CFS, the accumulationof fragments with a molecular mass of 37 kDa could be due toan increased proteolytic activity in PBMC extracts.

In summary, apart from recent infection, our findings provideadditional arguments to support the hypothesis that increasedproteolytic activity leads to the accumulation of a 37-kDa bindingpolypeptide in PBMC extracts from CFS patients and could beconsidered as a potential diagnostic marker. In the absenceof acute infection or chronic inflammation, a high RNase L isoformratio could distinguish CFS patients from healthy volunteers.Additional large studies and follow-up studies are requiredto confirm the stability of this high ratio of RNase L isoformin a CFS group.

ACKNOWLEDGMENTS

These studies have been supported by the Centre National dela Recherche Scientifique and by RED Laboratories (Brussels,Belgium). L.B. holds a postdoctoral position with RED Laboratories.

FOOTNOTES

* Corresponding author. Mailing address: Service de Médecine Interne, Hôpital Saint Antoine, 184 Rue du Faubourg Saint Antoine, 75571 Paris Cedex 12, France. Phone: 33 (1) 49 28 30 54. Fax: 33 (1) 1 49 29 28 85. E-mail: kiet.tiev{at}sat.ap-hop-paris.fr.

REFERENCES

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