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Winter 2000
RNase L: Hope for a
Biomarker
By Nancy L. Reichenbach,
Temple
University
Chronic
fatigue syndrome (CFS) presents a diagnostic challenge for physicians and patients as it is determined
through the application of a group of symptoms. Many of these symptoms are nonspecific and occur in other
illnesses that are characterized by fatigue, so it is often a lengthy and expensive process to eliminate
other clinical possibilities.
What is needed is an objective biological marker that can be used
as a definitive test. The measurement of the enzyme RNase L is one promising marker that is consistent
with an activated immune system in CFS.
The first studies of RNase L in individuals
with CFS were initiated because the clinical symptoms associated with CFS could often be explained by
a persistent viral infection or immune suppression, particularly in those patients who experience acute
onset. Viral infection of cells results in the production and secretion of cytokines, including the inter-ferons.
Interferons control the way cells respond to a virus through a group of inter-related enzymes that comprise
an antiviral defense pathway. This pathway is known as the 2',5'-oligoadenylate synthetase/RNase L pathway.
Antiviral pathway
abnormalities
The status of the
RNase L pathway is measured in humans by sampling peripheral blood mononuclear cells (lymphocytes). RNase
L is the key enzyme of the antiviral pathway, and it is designed to degrade viral RNA. While RNase L is
found in nearly all mammalian cells, it has to be "turned on" by a small molecule, 2-5A.
Binding
of 2-5A to RNase L changes the enzyme from its inactive (latent) state to its active state. When active,
RNase L inhibits viral protein synthesis and thereby prevents replication of a virus. However, overactive
RNase L can have detrimental effects on the body by degrading cellular RNA as well as viral RNA.
Several
critical parts of the antiviral defense pathway are not functioning correctly in many people with CFS.
The first evidence was obtained in a study of 15 people involved in what has been termed an "outbreak"
of CFS in Lake Tahoe, Nev. Thirteen of the 15 people studied had a dramatically increased (upregulated)
level of RNase L enzyme activity, as much as 1,500 times above normal levels1. As their symptoms
improved, their RNase L activity returned towards normal.
Confirmation of the effect of upregulated
RNase L was demonstrated in a four-city trial of individuals who were severely disabled by CFS. Blood
samples were obtained from individuals who fit the definition for CFS and from healthy individuals. Researchers
at Temple University School of Medicine demonstrated that the activity of the RNase L enzyme in lymphocytes
was significantly higher in the individuals with CFS than in the healthy controls2. And not
only was there an upregulation of RNase L activity in the CFS patients, there was a significant increase
in the level of 2-5A (the molecule that converts RNase L from its latent to its active state) and the
level of 2-5A synthetase (the enzyme that synthesizes the 2-5A activator molecule).
Perhaps
the most striking finding in lymphocytes from individuals with CFS is a unique form of the RNase L enzyme.
The size of the RNase L protein that is usually present in humans is 80 kilodaltons (kDa). However, in
many individuals with CFS, this 80 kDa enzyme is either scarce or missing altogether. A unique low molecular
weight (LMW) form of RNase L is observed instead3. Besides its smaller size (37 kDa), the LMW
RNase L has other biochemical differences from the 80 kDa RNase L. The LMW RNase L binds its activator
more tightly and is more potent than the 80 kDa form of RNase L.
A subsequent
large study of CFS patients revealed several connections between the RNase L pathway and clinical status4.
RNase L enzyme activity correlated well with the Metabolic Screening Questionnaire, a measure of general
health status. In addition, three measurements regarding the antiviral pathway increase (RNase L, bioactive
2-5A and the LMW RNase L) as the individual's ability to carry out the activities of daily living decreases,
indicated by a low Karnofsky Performance Score. Therefore, the increased activity of the RNase L pathway
is an indication of a lower state of general health. Current studies indicate that all three measurements
of the pathway are abnormal in individuals with CFS.
Confirmatory
research presented
The upregulation
of the RNase L pathway in CFS has been confirmed by at least four independent research studies. Researchers
from the University of Newcastle and University of Sydney in Australia have shown that elevated RNase
L activity is a good predictor of development of fatigue, muscle pain and reduced mood in individuals
with CFS5.
Other investigators have demonstrated that the mRNA and protein levels
of the RNase L inhibitor (RLI) are also decreased in individuals with CFS6. Decreased
level of RLI results in an accumu-lation of free RNase L and a concomitant increase in RNase L activity.
At
the Second World Congress on Chronic Fatigue Syndrome in Brussels in 1999, investigators from the Free
University of Brussels and Montpellier University presented research confirming the presence of the LMW
RNase L in CFS patients using a new biochemical method to detect the LMW and 80 kDa forms of RNase L.
They calculated a ratio of the LMW RNase L to the 80 kDa RNase L. Using a cut-off value of 0.5 for this
ratio, they found that CFS patients could be distinguished from healthy controls with very high accuracy.
More important, the RNase L ratio also distinguished individuals with CFS from individuals with fibromyalgia
or depression, two clinically similar illnesses to CFS7.
Results of another multi-disciplinary
study involving clinical and preclinical investigators from four countries (Belgium, France, Germany
and the United States) were also presented at the Brussels Congress. A study was designed to compare two
methods, which use different probes to detect RNase L in lymphocytes. With results expressed as a ratio
of the LMW 37 kDa RNase L protein compared to the 80 kDa RNase L, in a side-by-side comparison of coded
samples from CFS patients and controls, both methods accurately identified CFS patients8.
In
addition, this study indicates that the presence of the LMW RNase L is independent of duration of CFS
symptoms. The LMW RNase L was detected in individuals who had CFS symptoms for as long as 19 years.
The
cellular origin of the LMW RNase L is not known. It has been demonstrated that as the amount of the LMW
RNase L protein increases in lymphocytes, the amount of the 80 kDa RNase L protein decreases4.
In view of the com-plex changes in the immune response that have been demonstrated in CFS, it may be that
the LMW RNase L originated as part of the 80 kDa RNase L.
Purification and sequencing of the LMW
RNase L will be required to determine its origin, a daunting task because the protein is derived from
CFS patient lymphocytes. The abundance of the 80 kDa RNase L in mammalian cells has been estimated at
only 0.0005% of total cellular protein.
Potential for
a diagnostic test
The diagnosis of
CFS still relies heavily on the application of the CDC's symptom criteria, which has been useful in examining
the heterogeneous nature of the clinical presen-tation of CFS, but requires refinement. RNase L abnormalities
present a quantifiable, reproducible enzyme defect in individuals with CFS that is measurable by established
laboratory techniques.
RNase L testing has multiple potential applications. It has recently been
reported that RNase L protein levels can distinguish between CFS and two illnesses with similar clinical
presentations (fibro-myalgia and depression)7. The presence of the LMW RNase L identifies a
group of people with CFS who have an abnormally elevated antiviral response and therefore may respond
very well to antiviral therapies. In view of the association between RNase L and health status, measurement
of RNase L activity and protein level may help in the design of therapeutic approaches to CFS and as indicators
of response to such therapies.
RNase L protein level and enzyme activity are potentially
powerful diagnostic tools for the physician, when considered in conjunction with clinical evaluation and
other laboratory tests. These tests provide a means of identifying individuals with CFS by an objective
biochemical measurement.
Currently, the laboratory test for the LMW RNase L relies on preparation
of a lymphocyte pellet. When RNase L can be measured by immunoassay, it will be accessible to more physicians
and may find application as part of a CFS diagnostic test battery.
References
Suhadolnik RJ et al. Changes in the 2-5A
synthetase/ RNase
L antiviral pathway in a controlled clinical trial with poly(I) poly(C12U) in chronic fatigue syndrome.
In Vivo. 1994;8:599-604.
Suhadolnik RJ et al. Upregulation of the 2-5A synthetase/ RNase
L antiviral pathway associated with chronic fatigue syndrome. Clin Infect Dis.1994;18:S96-S104.
Suhadolnik RJ et al. Biochemical evidence for a novel low mole-cular
weight 2-5A-dependent RNase L in chronic fatigue syndrome. J Interferon & Cytokine Res.1997;17:377-85.
Suhadolnik RJ et al. Biochemical dysregulation of the 2-5A synthetase/RNase
L antiviral defense pathway in chronic fatigue syndrome. J Chronic Fatigue Syndrome.1999;5:223-242.
McGregor NR et al. The biochemistry of chronic pain and fatigue.
Second World Congress on Chronic Fatigue Syndrome and Related Disorders, Brussels, September 9-12, 1999.
Vojdani A, Choppa PC, Lapp CW. Downregulation of RNase L inhibitor
correlates with up-regulation of interferon-induced proteins (2-5A synthetase and RNase L) in patients
with chronic fatigue immune dysfunction syndrome. J Clin Lab Immunol. 1998;50:1-16.
De Meirleir K et al. A 37 kDa 2-5A binding protein as a potential
biochemical marker for chronic fatigue syndrome. Am J Med.2000;108(2):99-105.
Suhadolnik RJ et al. Diagnosis of chronic fatigue syndrome (CFS):
determination of a low molecular weight 37 kDa 2-5A dependent RNase L in peripheral blood mono-nuclear
cell extracts. Second World Congress on Chronic Fatigue Syndrome and Related Disorders, Brussels, September
9-12, 1999.
Ms. Reichenbach is an associate scientist
in the Department of Biochemistry
at Temple University School of Medicine, Philadelphia, Pa.
Individuals with CFS often
have:
- Upregulated RNase L activity
- Unique, low molecular weight form of RNase L
- Increased levels of 2-5A, the RNase L "activator" molecule
- Decreased levels of RNase L inhibitor
- Connection between clinical status and RNase L levels
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