Chronic fatigue syndrome diagnosis

Details Chronic fatigue syndrome diagnosis Chronic fatigue syndrome diagnosis

2000-04-13

2001-03-27

Schwartzman, Robert A. (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S007100, C435S007400, C435S007720, C435S018000

Reexamination Certificate

active

06207366

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the diagnosis of chronic fatigue syndrome (CFS) through detection and quantitation of RNase L molecules.
BACKGROUND OF THE INVENTION
CFS is an illness of unknown etiology, often associated with sudden onset, flu-like symptoms, debilitating fatigue, low-grade fever, myalgia and neurocognitive dysfunction. CFS patients typically display reduced Karnofsky performance scores (KPS). The Karnofsky performance test measures an individual's ability to function and carry on normal activities. Karnofsky scores range form zero for a dead patient to 100 for no evidence of disease. Diagnosis of CFS remains one of exclusion.
An accumulating body of evidence suggests that CFS is associated with disregulation of both humoral and cellular immunity, including mitogen response, reactivation of viruses, abnormal cytokine production, diminished natural killer cell function and changes in intermediary metabolites. It has been suggested that the clinical and immunological abnormalities observed in CFS might include defects in the double-stranded RNA (dsRNA)dependent, interferon-inducible pathways, i.e., the 2′,5′-oligoadenylate (2-5A) synthetase/RNase L and p68 kinase (PKR) antiviral defense pathways (Suhadolnik et al.,
Clin. Infect. Dis
. 18:S96-S104, 1994; Suhadolnik et al.,
In Vivo
8:599-604(1994). The 2-5A synthetase/RNase L pathway is part of the antiviral defense mechanism in mammalian cells; this pathway also has a role in the regulation of cell growth and differentiation (Lengyel,
Ann. Review Biochem
. 51:251-282, 1982; Sen et al.,
Adv. Virus Res
. 42:57-102, 1993).
When activated by dsRNA, 2-5A synthetase converts ATP to 2′,5′-linked oligoadenylateswith 5′-terminal phosphates. Biologically active 2-5A binds to and activates a latent endoribonuclease, RNase L, which hydrolyzes single-stranded viral and cellular RNA, primarily after UpNp sequences, thereby inhibiting protein synthesis.
Previous studies on the 2-5A synthetase/RNase L pathway in CFS revealed a statistically significant dysregulation in which the 2-5A synthetase is present predominantly in its activated form, bioactive 2-5A levels are elevated, and RNase L activity is upregulated (Suhadolnik et al.,
Clin. Infect. Dis
., supra; Suhadolnik et al.,
In Vivo
, supra). Expression of the serine-threonine kinase, PKR, is downregulated in CFS (Suhadolnik et al.,
In Vivo
, supra). PKR controls initiation of protein translation through phosphorylation of elF-2.
Despite these efforts, a clear cut molecular marker for CFS has not been identified. Diagnosis is presently carried out with resort to criteria recommended by the Centers for Disease Control and Prevention (Fukuda et al.,
Ann. Intem. Med
. 121:953-959, 1994). What is needed is a biochemical test, relying on an unambiguous molecular markers for CFS, which may form the basis of a definitive CFS diagnosis, or which may be employed as an adjunct to other CFS diagnostic methods.
Abbreviations
The following abbreviations may be used herein:
AMP: adenosine 5′-monophosphate;
2-Azido-AMP: 2-azidoadenosine 5′-monophosphate;
8-Azido-AMP: 8-azidoadenosine 5′-monophosphate;
2,8-Azido-AMP: 2,8-diazidoadenosine 5′-monophosphate;
2-5A: 2′,5′-oligoadenylate, that is, an oligomer of adenylic acid with (2′→5′)-phosphodiester linkages and 5′-riphosphate;
CFS: chronic fatigue syndrome;
dsRNA: double-stranded RNA;
ELISA: enzyme-linked immunosorbent assay;
etheno-AMP N
1
N
6
-ethenoadenosine 5′-monophosphate;
GST: glutathione S-transferase;
HEPES: 4-(2-hydroxyethyl)-piperazine ethanesulfonic acid;
PBMC: peripheral blood mononuclear cells;
PBS: phosphate-buffered saline;
p
3
A
3
trimer of adenylic acid with (2′→5′)-phosphodiester linkages and 5′-triphosphate;
pApAp(8-azidoA): 5′-O-phosphoryl-adenylyl-(2′→5′)-adenylyl-(2′→5′)-8-azidoadenosine;
poly(U)-3′-pCp: polyuridylic acid having a cytosine residue attached to the 3′terminus thereof;
SDS-PAGE: sodium dodecylsulfate-polyacrylamide gel electrophoresis.
SUMMARY OF THE INVENTION
A method fordiagnosing chronicfatigue syndrome comprises
determining the level of low molecular weight RNase L and the level of high molecular weight RNase L in a biological sample from a human subject; and
computing a ratio for the level of low molecular weight RNase L to the level of high molecular weight RNase L, which ratio is diagnostic for chronic fatigue syndrome.
According to an embodiment of the invention, a ratio of low molecular weight RNase L to high molecular weight RNase L of greater than 0.15 indicates that the individual is afflicted with chronic fatigue syndrome.
The biological sample preferably comprises blood or a fraction thereof, most preferably a cytoplasmic extract of peripheral blood mononuclear cells.
According to one embodiment of the invention, the level of the low and high molecular weight RNase L are determined by
(a) contacting the sample with a probe comprising a compound according to formula I bearing a detectable label, under conditions sufficient to form covalent conjugates of the labeled probe compound and 2′,5′-oligoadenylate-binding proteins in the sample:
wherein
m is an integer from 0 to 3,
n is an integer from 1 to 3, and
each R
1
and each R
2
is, independently of each other R
1
and R
2
, hydrogen or N
3
, provided at least one R
1
or R
2
is N
3
, or a water soluble salt of said compound;
(b) contacting the sample containing the covalent conjugates with an antibody which binds RNase L species in the sample;
(c) separating the proteins in the sample by gel electrophoresis; and
(d) quantitating the proteins of about 37 kDa and about 80 kDa apparent molecular weight according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which about 37 kDa and about 80 kDa proteins have formed covalent conjugates with the labeled probe compound and which are bound by the antibody.
By “about” with reference to the molecular weight of the various RNase L forms is meant a molecular weight with the range of plus or minus 3 kDa.
DETAILED DESCRIPTION OF THE INVENTION
Biologically active 2-5A binds to and activates the latent endoribonuclease, RNase L. Activated RNase L in turn hydrolyzes single-stranded viral and cellular RNA, thereby inhibiting protein synthesis. Cell extracts from healthy individuals contain an about 80 kDa 2-5A binding protein (hereinafter “high molecular weight RNase L”) with 2-5A-dependent RNase L activity. CFS patients, on the other hand, have been demonstrated to possess a distinct about 30 kDa 2-5A binding protein having 2-5A-dependent RNase L activity (hereinafter “low molecular weight R Nase L”). By “RNase L activity” is meant the enzymatic activity of RNase L in hydrolyzing its RNA substrates. The 30 kDa RNase L identifiable in cell extracts from individuals with CFS has the same 2-5A binding function as the 80 kDa RNase L and exhibits 2-5A-dependent activity similar to that of the 80 kDa RNase L.
The low molecular weight RNase L has a molecular weight of about 30 kDa under native conditions. Essentially, native conditions are conditions which do not denature proteins, most particularly, conditions which do not denature enzymes. Under the denaturing conditions of SDS-PAGE, low molecular weight RNase L has an apparent molecular weight of about 37 kDa. The 37 kDa mass under denaturing conditions upon SDS-PAGE analysis is in reasonable agreement with the 30 kDa mass under native conditions, based on literature precedents accounting for differences in molecular mass observed under denaturing and native conditions (Somerville etal.,
J. Bactedol
. 117:3837-3842, 1995). As used herein, the expressions “30 kDa RNase L”, “37 kDa RNase L” and “low molecular weight RNase L” all mean the same molecule, and are used interchangeably herein. As used herein, the expressions “80 kDa RNase L” and “high molecular weight RNase L” mean the same molecule, and are used interchangeably herein.
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