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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Neuromuscul Disord. Author manuscript; available in PMC Mar 1, 2011.
Published in final edited form as:
PMCID: PMC2841214
NIHMSID: NIHMS174616

Autoantibody Profiles in Two Patients with Non-Autoimmune Muscle Disease Implicate a Role for Gliadin Autoreactivity

Abstract

The objective of this case study was to characterize autoreactivity in two patients with non-autoimmune forms of muscle disease who had positivity for antinuclear antibodies (ANA) and Ro (SSA) autoantibodies. Serum samples from these two patients were applied to an autoantigen protein array with more than 70 specificities and were compared to samples from healthy controls and patients with systemic lupus erythematosus. Both myopathy patients had high levels of gliadin autoreactivity in serum and one patient had an overall autoantibody profile with lupus-like features. The findings suggest that some disorders of muscle that are considered nonautoimmune, may in fact have autoimmune features. Further examination of the role of subclinical gluten autoreactivity in the pathogenesis of myopathy syndromes has the potential to suggest improved approaches to diagnosis and treatment of these conditions.

Keywords: autoantibodies, C1q, dysferlinopathy, gliadin, myopathy

Inflammatory muscle disease occurring in the absence of cutaneous features presents major diagnostic challenges. The Bohan and Peter diagnostic criteria for these disorders that have been used for over 30 years reliably identify individuals with dermatomyositis (DM), but specificity for polymyositis (PM) is low [1;2]. Furthermore, greater complexities have been recognized since the criteria were originally developed, notably the added disease of inclusion body myositis (IBM). IBM is relatively resistant to immunosuppressives, so making a distinction between PM and IBM has major implications for treatment. Other muscle disorders such as dystrophies also have been recognized to have diverse presentations and findings that can be misdiagnosed as PM using the Bohan and Peter criteria. In fact, it has been suggested that pure PM is a rare entity that is over-diagnosed [3].

A muscle biopsy is required to make a definitive diagnosis of PM, but the inflammatory changes can be discontinuous, so even this test can produce inconclusive results. Given these difficulties, when the clinical evaluation uncovers serum autoantibodies, an immune-mediated process is suspected and this is likely to influence treatment choices. We report here two cases of patients with biopsy-documented non-inflammatory muscle diseases in whom detection of serum autoantibodies including antinuclear antibodies (ANA) and Ro/SSA led to further diagnostic evaluations and in one case supported several futile courses of immunosuppressive therapies for presumed PM. Further study of an expanded array of autoantibody profiles in these two patients revealed high levels of reactivity with gliadin, an autoantigen which has been associated with inflammatory muscle diseases and IBM. The findings in our two patients suggest that autoreactivity in general and anti-gliadin antibodies in particular might be associated with muscle disorders, including some that are not currently considered autoimmune.

Case Report

Patients

Patient 1 is an African-American female who first noted the gradual onset of muscle weakness when she was 20 years old. A muscle biopsy was interpreted as being consistent with an idiopathic inflammatory myopathy (IIM). She was treated with glucocorticoids, but had increasing weakness and presented to our facility the following year for evaluation. She had no other significant past history. Family history was positive for rheumatoid arthritis in a grandmother. The patient was an only child; she did not use tobacco or alcohol. Examination confirmed significant weakness in a predominantly proximal distribution. No rash or skin abnormalities were noted. She had borderline tachycardia (HR 101 bpm), elevated CK with a maximum value of 37,560 U/L (normal 30-135 U/L) and low serum creatinine of 0.3 mg/dL (normal 0.6-1.2 mg/dL). The test for antinuclear antibodies (ANA) was positive at 145 Elisa Units (normal < 20 EU) and Ro (SSA) antibodies were also positive. EMG showed low-amplitude motor units with increased recruitment pattern and other changes consistent with muscle inflammation. She subsequently had increased weakness despite treatment with methotrexate, azathioprine and infliximab, so that she required a walker, and then a wheelchair. CK values remained elevated (3000 to 12,000 U/L). A repeat muscle biopsy was interpreted as showing chronic myopathic changes and she was told that she had mixed connective tissue disease (MCTD). She received intravenous immunoglobulin monthly, mycophenolate mofetil (2500 mg daily), and prednisone, (20 mg daily). Her activities were severely limited so that she became largely bed-bound. She was subsequently hospitalized with pneumonia, and a third muscle biopsy performed during this hospitalization revealed chronic myopathic alterations associated with attenuated sarcolemmal dysferlin reactivity, consistent with limb girdle muscular dystrophy 2B (dysferlinopathy; Figure 1A). Subsequently, immunosuppressive medications were discontinued on hospital discharge, and steroids were tapered off over the next two months. She became ventilator-dependent, required intermittent institutional care and expired outside of the hospital due to respiratory failure more than 6 months later. Unfortunately, a whole blood sample sent for dysferlin measurement in monocytes was judged to be of insufficient quality for assay by the outside laboratory, and the patient expired before a repeat sample could be obtained. Genetic testing was not possible due to cost and consent issues.

Figure 1
Quadriceps muscle biopsy from Patient 1. The H&E stain (panel A, left) reveals abnormal fiber size variability, accompanied by a spectrum of chronic myopathic changes including occasional degenerating and regenerating fibers and mildly increased ...

Patient 2 is a 49 year old white female with a 3-year history of muscle weakness and balance problems who presented on referral from a neurologist for consultation regarding a positive ANA. She noted trouble walking, muscle spasms and numbness that were worse in the lower extremities than in the upper extremities. Neurologic evaluation had documented decreased proprioception and vibratory sensation in the lower extremities. She began to use a cane and eventually needed a wheelchair to travel outside of her home. She denied any skin problems, dryness of eyes or mouth and had no trouble swallowing. Past history was positive for bulimia and chronic renal insufficiency with a creatinine of 3.5 mg/dL that was thought related to the past use of nonsteroidal anti-inflammatory drugs. Family history was positive for breast cancer in a sister, heart disease in her father and Parkinson’s disease in her mother. The patient had worked as a nurse. She did not use tobacco or alcohol. Testing for transthyretin (TTR) gene abnormalities associated with familial amyloidotic polyneuropathy was negative as were autoantibodies to Ri that may be seen in paraneoplastic syndromes; sulfatide and Hu autoantibodies were also absent (testing done at Athena Diagnostics, Worcester MA). On exam, muscles of the extremities were decreased in bulk and she was unable to lift either leg against gravity; grip strength was reduced bilaterally. Deep tendon reflexes were increased in the upper extremities and decreased in the lower extremities; she had no clonus or loss of sensation. Laboratory tests included ANA >1:2560 with a homogeneous pattern (confirmed as154 Elisa Units). The ENA panel was positive for SSA (Ro) and negative for SSB (La) and all of the other panel autoantibodies, including Jo-1. The sedimentation rate and CK were within normal ranges. A quadriceps muscle biopsy showed desmin-reactive hyaline bodies in the sarcoplasm of multiple myofibers, compatible with myofibrillar myopathy [4] (Figure 2). Intermittent prednisone treatment resulted in slight improvement, but she remained very weak.

Figure 2
Quadriceps muscle biopsy from Patient 2. The H&E stain (panel A, top left) shows fiber size variability, accompanied by the presence of well-developed sarcoplasmic hyaline inclusions in a significant minority of myofibers (arrows). The inclusions ...

Both patients were consented for enrollment in the Dallas Regional Autoimmune Disease Registry (DRADR), which is a research registry developed for the study of autoimmunity and autoimmune diseases [5] and they provided serum samples that were stored at −80 degrees C. Autoantibody studies included samples from two comparison groups drawn from individuals in the DRADR: 7 females with systemic lupus erythematosus (SLE) and 7 healthy control (HC) females without family history of autoimmune disease. Analyses of the serum samples for IgM, IgG and IgA autoantibodies were carried out on a protein array composed of more than 70 autoantigens described previously[6]. Results were expressed as mean fluorescence units (MFU). The array data were analyzed using the Cluster and Treeview software of Michael Eisen (http://rana.lbl.gov/eisen/).

Clustering analysis of the IgG autoantibodies showed two major groups: one with only HCs and the other with all of the SLE patients. The two MD patients clustered separately, suggesting different relatedness to SLE, with Patient 1 who had been thought to have had MCTD being more SLE-like (Figure 3). The mean values for MFU in each of the three diagnostic groups were used to determine which autoantigens showed the greatest differences between the MD patients and the other two groups while also showing similar results for the two MD patients. This analysis showed that IgG and IgA anti-gliadin were higher in both MD patients than in all but one of the 7 SLE patients (Figure 3). The related IgG autoantibody to transglutaminase 2 (TG2) was elevated in MD Patient 1 (178 MFU vs. mean value of 12 MFU in HC group) and also in one SLE patient (384 MFU) who did not have concomitant elevation of gliadin autoantibodies. IgG and IgM antibodies to C1q were also elevated in both MD patients; IgG antibodies to C1q and gliadin were highly correlated (R2=0.946; P<0.0001). No other antibodies had correlation coefficients with C1q of greater than 0.7. Reactivity to the 60kD Ro autoantigen was present in both MD patients, consistent with the clinical laboratory findings. IgG antibodies to the muscle-related protein myosin were lower in the two MD patients than in either the HC or SLE groups, suggesting that autoreactivity to this component of damaged muscle was not present.

Figure 3
Clustering analysis of IgG autoantibody profiles measured on the autoantigen array in 7 healthy controls (HC), 7 SLE patients and 2 muscle disease (MD) patients (left panel). Each row represents one autoantibody specificity and each column represents ...

Discussion

The two patients presented in this report had muscle problems that are not considered autoimmune. Patient 1 had limb-girdle muscular dystrophy, a disease with a highly variable clinical phenotype, ranging from asymptomatic patients who only have high levels of muscle enzymes to those with severe weakness. In one series, 10 of 40 of these patients (25%) were misdiagnosed as having PM and this subset had more rapid disease progression, similar to the course in our patient [7]. Respiratory failure as in our patient is unusual in this syndrome and it is also not observed in lupus-related disorders. Unfortunately detailed genetic testing needed to confirm the diagnosis could not be carried out in this case and it is possible that an unusual genetic variant of this highly variable disease was present. Autoantibody testing has not been previously reported in this disease, but a case in which ANA positivity along with a muscle biopsy showing inflammation led to a mistaken diagnosis of PM has been described [8]. The myopathy in Patient 2 is less well-defined pathologically but resembles the syndrome of hyaline masses and nemaline rods recently described in familial cardioneuromyopathy [4]. Autoantibody studies have not been reported in this syndrome.

We were interested in the high levels of gliadin autoreactivity detected on the autoantigen array because an association between antibodies to gliadin and neuromuscular disease has been previously described. In one study, anti-gliadin antibodies were present in 17 out of 51 (31%) patients with inflammatory myopathies, including all 4 patients with sporadic IBM [9]. Two of these 4 IBM patients also had antibodies to Ro. None of the patients had TG2 autoantibodies; reasons for the greater prevalence of gliadin autoantibodies were not clear. Three of 5 individuals with gliadin autoreactivity had small bowel biopsy changes that were consistent with celiac disease (CD). Treatment with a gluten-free diet contributed to clinical improvement in one DM patient, but not in the two IBM patients. This finding is consistent with other data indicating that subclinical CD may be more common than previously thought; the prevalence might be as high as 1% [10].

Autoimmune disease in the setting of LGMD2B has been reported previously in a patient who had autoimmune adrenal insufficiency and sarcoidosis along with dysferlin deficiency [11]. Of further interest is the observation that dysferlin-deficient monocytes show enhanced phagocytic properties [12] and that altered vesicular trafficking in muscles of these patients creates a local inflammatory environment [13]. Taken together with the present findings, the evidence suggests that further studies of immune system function in dysferlin deficient patients would be of interest.

The reason for Ro autoreactivity in our patients and in other myopathies is unknown, although muscle tissues do show high levels of expression of this protein [14]. Whether muscle damage exposes this autoantigen thus inciting an antibody response is one possibility. The high correlation between antibodies to gliadin and C1q was unexpected; one possibility is that this relates to molecular similarities in the molecules, both of which contain long and repetitive amino acid sequences.

The findings in our two patients suggest that further studies of autoantibody profiles in patients with various forms of muscle disease, both inflammatory and non-inflammatory, would be of interest. Whether gluten-withdrawal treatment trials in these patients would be useful is controversial, and would be complicated by the need for duodenal biopsies since definitive serologic tests are as yet not available [15].

Acknowledgements

This work was supported in part by NIH P50 AR 055503. NJO receives support from the McGee Foundation and HP was supported by the Doris Duke Foundation.

Footnotes

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