Chronic fatigue syndrome diagnosis

Details Chronic fatigue syndrome diagnosis Chronic fatigue syndrome diagnosis

1999-04-21

2001-04-10

Gitomer, Ralph (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S018000

Reexamination Certificate

active

06214554

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the diagnosis of chronic fatigue syndrome (CFS) through detection of a unique molecular marker associated with the disease.
BACKGROUND OF THE INVENTION
Chronic fatigue syndrome (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 nonfunctional or dead patient to 100 for a completely normal function. 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 oligoadenylates with 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 eIF-2.
Despite these efforts, a clear cut molecular marker for CFS has not been identified. What is needed is a biochemical test, relying on an unambiguous molecular marker for CFS, which may form the basis of a definitive CFS diagnosis.
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′-triphosphate;
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 for diagnosing chronic fatigue syndrome comprises assaying a patient sample for the presence of an about 30 kDa RNase L.
According to one embodiment, the method comprises (a) fractionating the proteins in the patient sample according to molecular weight under nondenaturing conditions, and (b) assaying the fractionated proteins for an about 30 kDa protein having RNase L activity. The RNase L activity assay may comprise, for example, detecting formation of specific cleavage products (SCP) from hydrolysis of 28S and/or 18S RNA, or detecting the hydrolysis of poly(U)-3′-pCp.
According to another embodiment, the invention includes a method for diagnosing chronic fatigue syndrome comprising:
(a) contacting a patient sample prepared in the presence of added protease inhibitor with a probe comprising a compound according to formula I bearing a detectable label, under conditions sufficient to form covalent conjugates of said 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 said covalent conjugates with an antibody which binds RNase L species in the sample;
(c) separating the proteins in said sample by gel electrophoresis; and
(d) examining said separated proteins for marker proteins which
(i) have formed a covalent conjugate with the labeled probe compound, and
(ii) are bound by said antibody;
the presence of a marker protein of about 37 kDa apparent molecular weight according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis being diagnostic for chronic fatigue syndrome.
According to yet another embodiment, the invention includes a method for detecting severe chronic fatigue syndrome comprising:
(a) contacting a patient sample with a probe comprising a compound according to formula I bearing a detectable label, under conditions sufficient to form covalent conjugates of said labeled probe 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 said covalent conjugates with an antibody which binds RNase L species in the sample;
(c) separating the proteins in said sample by gel electrophoresis; and
(d) examining said separated proteins for marker proteins which
(i) have formed a covalent conjugate with the labeled probe compound, and
(ii) are bound by said antibody;
the presence of a marker protein of about 37 kDa apparent molecular weight according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the absence of a marker protein of about 80 kDa apparent molecular weight according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being diagnostic for severe chronic fatigue syndrome.
According to a further embodiment of the invention, a method for assessing the relative severity of chronic fatigue syndrome is provided comprising assaying a patient sample under native conditions for the presence of RNase L molecules having molecular weights of about 30 and about 80 kDa. The presence of an about 30 kDa RNase L molecule, and the absence of an about 80 kDa RNase L molecule, indicates severe chronic fatigue syndrome. The presence of RNase L molecules of both molecular weights indicates less severe chronic fatigue syndrome. The assay may be conducted by (a) fractionating the proteins in the patient sample according to molecular weight under nondenaturing conditions; and (b) assaying the fractionated proteins for about 30 kDa and about 80 kDa proteins having RNase L activity.


REFERENCES:
patent: 4990498 (1991-02-01), Suhadolnik
patent: 5258369 (1993-11-01), Carter
patent: 5776690 (

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