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Rheumatology 2001; 40: 806-810
© 2001 British Society for Rheumatology

Original Papers

Autoantibodies to a 68/48 kDa protein in chronic fatigue syndrome and primary fibromyalgia: a possible marker for hypersomnia and cognitive disorders

M. Nishikai, S. Tomomatsu1, R. W. Hankins1, S. Takagi, K. Miyachi2, S. Kosaka and K. Akiya

National Tokyo Medical Center, Tokyo,
1 Health Sciences Research Institute, Yokohama and
2 Keigu Medical Clinic, Yokohama, Japan


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To identify antinuclear antibodies (ANA) specific for chronic fatigue syndrome (CFS), and in related conditions such as fibromyalgia (FM) or psychiatric disorders.

Methods. One hundred and fourteen CFS patients and 125 primary and secondary FM patients were selected based on criteria advocated by the Centers for Disease Control and Prevention and by the American College of Rheumatology, respectively. As controls, healthy subjects and patients with either various psychiatric disorders or diffuse connective tissue diseases were included. Autoantibodies were examined by immunoblot utilizing HeLa cell extracts as the antigen.

Results. Autoantibodies to a 68/48 kDa protein were present in 13.2 and 15.6% of patients with CFS and primary FM, respectively. In addition, autoantibodies to a 45 kDa protein were found in 37.1 and 21.6% of the patients with secondary FM and psychiatric disorders, respectively. Meanwhile, these two autoantibodies were not found at all in connective tissue disease patients without FM, nor in healthy subjects (P<0.05). As a group, the anti-68/48 kDa-positive CFS patients presented more frequently with hypersomnia (P<0.005), short-term amnesia (P<0.07) or difficulty in concentration (P<0.05) than those CFS patients without the antibodies.

Conclusions. The presence of the anti-68/48 kDa protein antibodies in a portion of both CFS and primary FM patients suggests the existence of a common immunological background. These antibodies may find utility as possible markers for a clinicoserological subset of CFS/FM patients with hypersomnia and cognitive complaints.

KEY WORDS: Anti-68/48 kDa protein antibodies, Anti-45 kDa protein antibodies, Chronic fatigue syndrome, Fibromyalgia, Antinuclear antibodies, Sleep disorders, Hypersomnia, Insomnia, Depression, Neurosis.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Chronic fatigue syndrome (CFS) is a disease characterized by debilitating fatigue lasting for more than 6 months, in which generalized myalgia, sleep disorders, headache, depression, cognitive disorders and sicca symptoms are also found [1–3]. Although there does not currently exist a specific diagnostic test for CFS, the presence of antinuclear antibodies (ANA) has been reported with increasing frequency in CFS patients in contrast to that observed in healthy subjects [4–6]. Recently, ANAs to specific protein antigens have been reported in the sera of adult [7, 8] and juvenile [9] CFS patients. However, it has not been properly addressed whether these ANAs are associated with specific symptoms or with other disease conditions. It is felt that clarification of such uncertainties could lead to a reclassification of CFS and other patients into subsets based not only on clinical features, but on immunological characteristics as well.

Thus, the goals of the present study were as follows: (1) to determine the prevalence of ANAs to specific antigens in adult Japanese patients with CFS and CFS-related diseases such as primary fibromyalgia (FM, formerly termed fibrositis), major depression, or other psychiatric disorders, and (2) to establish correlations between any of these autoantibodies and specific clinical features.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Patients
One hundred and fourteen patients with CFS and 125 patients with FM were identified by one of the authors (MN) at the rheumatology out-patient clinic of the Department of Internal Medicine at the National Tokyo Medical Center during the 10-yr period between 1991 and 2000. Diagnoses were made according to the classification criteria advocated by the Centers for Disease Control and Prevention for CFS [2] and by the American College of Rheumatology for FM [10]. Sixty-four of the 114 CFS patients (56.1%) also met the FM criteria. The 125 FM patients were divided into 90 primary FM and 35 secondary FM patients. Among the 35 secondary FM patients, 13 patients also presented with systemic lupus erythematosus (SLE), nine with rheumatoid arthritis (RA), four with hepatitis C, three with CREST syndrome (a variant of systemic sclerosis), two each with Sjögren's syndrome and hypothyroidism, and one each with Behçet's disease, adult Still's disease, hepatitis B, and chronic nephritis. As control groups, 37 patients with psychiatric disorders (15 with major depression, seven with neurosis, and 15 with schizophrenia), 19 patients with diffuse connective tissue diseases (10 with SLE and nine with RA), and 37 healthy subjects matched for mean age, sex ratio and race with the 114 CFS patients were selected. Patients with psychiatric disorders were diagnosed by a psychiatrist (ST) in the Department of Psychiatry at the National Tokyo Medical Center. In order to determine the presence of any clinical features according to severity of depression, Beck's depression inventory [11] was used to divide the patients into those responding positively to less than 21 items on the questionnaire from those responding to 21 or more items. To characterize personality disorders of CFS patients, the structured clinical interview of DSM-III-R [12] was used. SLE and RA were diagnosed by criteria put forth by the American Rheumatism Association [13, 14]. Statistical comparisons were made not only between the CFS/FM patient groups and healthy controls, but also among the various patient groups as well.

Immunofluorescence microscopy
ANA were evaluated by the standard indirect immunofluorescence technique using HEp-2 cells (Medical and Biological Laboratories, Nagoya, Japan) as the substrate [6]. Patients were considered to be positive for ANA if immunofluorescent staining was observed at a serum dilution of 1:40.

Enzyme-linked immunosorbent assay (ELISA)
Autoantibodies to double-stranded deoxyribonucleic acid (dsDNA) and to extractable nuclear antigens such as Sm, U1 RNP, Ro(SSA), La(SSB) and Scl-70 (topoisomerase I) were tested by ELISA.

Double immunodiffusion
Screening tests for precipitating autoantibodies to the extractable nuclear and cytoplasmic antigens, including the Jo-1 and Mi-2 antigens, were carried out by double immunodiffusion [15] using a kit (Medical and Biological Laboratories).

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting
Antigens were extracted with NET-2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% NP-40) from nuclei of proliferating HeLa cells isolated following ultrasonication. SAD–PAGE was performed according to the method of Laemmli [16] using a minigel electrophoresis apparatus (Atto, Tokyo, Japan). Antigens were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Atto) at 100 mA per membrane for 90 min using a semidry blotting apparatus (Nihon Eido, Tokyo, Japan).

Proteinase K treatment
In order to verify the protein identity of the 68/48 kDa antigen, proteinase K-treated HeLa cell extracts were used in a portion of the SDS–PAGE/immunoblotting experiments.

Statistical methods
Fisher's exact probability test was used in the 2x2 tables. Differences in the means were analysed by Mann–Whitney's U-test.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
The demographics of the various patient groups are shown in Table 1Go. The male:female ratio did not differ among the groups. However, the mean ages of patients with secondary FM and psychiatric diseases were higher than those of CFS and primary FM patients (P<0.001).


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  		TABLE 1. Demographic background of the Japanese patients studied

 
Immunoblot experiments revealed the presence of autoantibodies with various antigenic specificities among the CFS patients. In particular, antibodies to a 68/48 kDa antigen were found most frequently among patients with CFS and primary FM (Fig. 1Go, Table 2Go). The prevalences of these antibodies in the two diseases were significantly higher when compared with healthy subjects (P<0.05) for both comparisons) and with connective tissue diseases (P<0.05 for both comparisons). On the other hand, these anti-68/48 kDa autoantibodies were found among the various psychiatric diseases at a very low frequency, not significantly different from healthy subjects (P=0.2397). These antibodies were not found in patients with non-FM diffuse connective tissue diseases, nor in healthy subjects.



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  		FIG. 1. Immunoblot analysis of autoantibodies to the 68/48 kDa protein and the 45 kDa protein found in CFS/FM and major depression. Lane 1, marker proteins for molecular weights; lanes 2, 3, CFS patients without FM; lanes 4, 5, CFS patients with FM; lanes 6, 7, primary FM without CFS; lanes 8, 9, major depression; lane 10, healthy subjects.

 

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  		TABLE 2. Prevalence of the autoantibodies to the 68/48 kDa proteins in CFS/primary FM and other conditions

 
The second most frequently observed autoantibody was that against a 45 kDa antigen (Fig. 1Go). This anti-45 kDa antibody was found in 37.1 and 21.6% of patients with secondary FM and psychiatric diseases, respectively. Each of these prevalences was significantly higher when compared against that of healthy subjects and those of other patient groups (Table 3Go). Among the 35 secondary FM patients, the anti-45 kDa antibody was found most frequently in the SLE subgroup (8 of 13, 62%) and not at all in the RA subgroup.


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  		TABLE 3. Prevalence of autoantibodies to the 45 kDa protein in patients with CFS/primary FM and other conditions

 
All immunoblot reactivities against the 68/48 kDa and the 45 kDa antigens were lost following pre-treatment of the HeLa cell extract with proteinase K, indicating that the anti-68/48 kDa and anti-45 kDa antibodies were being directed against protein components (data not shown).

The distributions of clinical features listed in the original [1] and revised [2] diagnostic criteria for CFS were compared between CFS patients with and without the anti-68/48 kDa antibody (Table 4Go). Increased frequencies of hypersomnia, impaired memory (short-term amnesia) and difficulty in concentration were observed in patients with these antibodies.


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  		TABLE 4. Clinical and laboratory characteristics of the CFS patients with and without the anti-68/48 kDa protein antibodies

 
ANAs as tested by indirect immunofluorescence were positive in all but one of the 15 anti-68/48 kDa-positive CFS patients with the common ‘diffuse/speckled’ staining pattern [6]. Of the 114 CFS patients, only three presented with precipitating antibodies, all of whom also tested positive for the anti-68/48 kDa antibodies. Antigenic specificities of these antibodies appeared to be different from any of the currently known types of precipitating antibodies such as Sm, U1 RNP, Ro, La, ribosomal P, Scl-70 (topoisomerase I), Jo-1 or Mi-2. The remaining 12 of the 15 anti-68/48 kDa-positive CFS patients did not have precipitating antibodies.


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
ANAs characteristic for CFS patients have been reported as being specific for nuclear membranes [7, 8]. In the present study, nuclear membrane immunofluorescent staining patterns were not observed among any of the Japanese CFS patients, despite using the same HEp-2 cells as the substrate [6]. In addition, the cognate antigen has been reported previously as a single 68 kDa protein, putatively thought to be the lamin B component of the nuclear membrane structure [7]. Our results with the immunoblot using the same HeLa cells confirmed the presence of antibodies not only against this 68 kDa protein, but also against a 48 kDa protein as well. This suggests that CFS patients can be divided into serologically distinct subsets. Furthermore, the similar prevalences of anti-68/48 kDa antibodies among primary FM patients and CFS patients raises the possibility that these two patient groups may share a common immunological abnormality. Drawing similarities between CFS and primary FM remains controversial [17–19], but our results suggest that these two diseases are similar not only clinically but also serologically.

The CFS patients with anti-68/48 kDa antibodies presented more frequently with hypersomnia and cognitive disorders, such as impaired memory (short-term amnesia) and difficulty in concentration, were frequently observed. It is possible that the cognitive disorders in the CFS patients may be the result of sleep disorders. It is of note that CFS patients without these antibodies presented with insomnia at a significantly higher frequency (Table 4Go). Because hypersomnia and insomnia have been classified as different sleep disorders, possibly with separate aetiologies [11, 20], it is felt that these autoantibodies may function as potential mediators for such disorders. Future studies are indicated to clarify the possible association of insomnia with immunological disorders. Although the 1994 revised diagnostic criteria for CFS [2] referred to ‘unrefreshing sleep’ as a term for defining sleep disorders, previous descriptions of insomnia and hypersomnia might be more appropriate in light of our data.

At present, it is not known whether these concomitant disorders are autoantibody mediated. It has only been in recent years that the significance of immunological abnormalities in primary sleep disorders has been studied [21–23]. The association of rapid eye movement sleep disorders with class II human leucocyte antigen (HLA) DQwl [24] raises the possibility that certain sleep disorders may arise from autoimmune mechanisms. It should be mentioned, however, that anti-locus ceruleus antibodies have not been found under conditions of severe monoaminergic cell loss in the locus ceruleus of patients with sleep disorders [25].

Elsewhere, in terms of elucidating the association with memory disorders, it remains to be seen whether the cognate antigen(s) of anti-68/48 kDa antibodies are related to any particular component of the hippocampus or amygdala which are known as the centres for short-term memory.

Finally, an autoantibody to a 45 kDa protein was newly identified as a possible marker for secondary FM and certain psychiatric diseases. The existence of an immunological difference between primary and secondary FM was quite unexpected as the two groups are quite similar in their clinical features [10, 26]. The anti-45 kDa antibody was found primarily in secondary FM patients with SLE. However, it is felt that this antibody is not SLE related, as known marker autoantibodies for SLE such as those against dsDNA, Sm, Ro(SSA) and La(SSB) were found with similar frequencies in both anti-45 kDa-positive and anti-45 kDa-negative SLE patient groups. In addition, the anti-45 kDa antibodies were found in three secondary FM patients without SLE, two with hepatitis C, one with adult-onset Still's disease and in a number of patients with various psychiatric diseases. It is of note that this anti-45 kDa antibody was found in secondary FM patients with SLE, but not in those with RA. Thus, separate immunological backgrounds may exist for the two secondary FM subgroups.

Positive ANA have been reported among patients with major depression [27, 28]. In this study, anti-45 kDa antibodies were also found in 22% of in-patients with psychiatric diseases and this prevalence was significantly higher (P<0.0001) when compared with the 3% prevalence observed in CFS patients. This relationship is opposite to that observed for anti-68/48 kDa antibodies between the two groups. It is of interest to note that the anti-45 kDa antibody was found among patients with neurosis, depression and schizophrenia with frequencies of 29, 20 and 20%, respectively. Thus, although these three psychiatric disorders are firmly established as clinically discrete entities, a portion of such patients may share a common immunological background, the precise aetiological role of which remains to be elucidated.

In this report, two new autoantibodies, the anti-68/48 kDa and the anti-45 kDa, have been described as possible markers for certain clinical subsets of CFS/primary/FM and of secondary FM/psychiatric disorders, respectively. In particular, the anti-68/48 kDa antibodies are considered to be closely associated with CFS/FM patients presenting with hypersomnia and/or cognitive disorders.


   Notes
 
Correspondence to: M. Nishikai, Department of Internal Medicine, National Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro-ku, Tokyo 152-8902, Japan. Back


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 2 October 2000; Accepted 16 February 2001


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