Striatal antibodies in children with Tourette's syndrome: multivariate discriminant analysis of IgG repertoires
Introduction
Gilles de la Tourette syndrome (TS) is a neuropsychiatric disorder characterized by the presence of chronic motor and vocal tics. The pathogenesis of TS remains unclear, although complex genetic and environmental factors appear to be involved. Evidence suggests that TS is an inherited disorder in which environmental (nongenetic) factors either have an etiologic role or mediate the form or severity of the phenotype (Walkup et al., 1996). Several environmental factors have been suggested as possible modifying agents, including prenatal events, exposure to drugs, and preceding infections Leckman et al., 1987, Leckman et al., 1990, Leckman and Peterson, 1993, Peterson et al., 1992, Sanchez-Ramos and Weiner, 1993 Clinical observations have associated recent infection with the abrupt onset of tics Kiessling, 1989, Kiessling et al., 1993, Kondo and Kabasawa, 1978, and studies have identified serum antibodies that cross-react with brain tissue. Antineuronal antibodies, determined by both immunofluorescent staining and ELISA methods, have been identified in children with neurobehavioral problems and movement disorders (tics, TS, and chorea), more often than in control children Kiessling et al., 1993, Kiessling et al., 1994, Laurino et al., 1997, Muller et al., 2000, Singer et al., 1998, Singer et al., 1999. A therapeutic response to plasma exchange and intravenous gamma globulin in selected patients also suggests that antineuronal antibodies may play a role in TS pathology (Perlmutter et al., 1999).
Western blotting has surfaced as a powerful tool for detecting specific autoantibodies in immune diseases. Complicating the straightforward identification of pathogenically relevant antigens, however, is that normal sera contain large amounts of natural antibodies which manifest themselves in complex staining patterns Avrameas, 1991, Haury et al., 1994, Zimmermann, 1993, Zimmermann and Weiss, 1987. Thus, detecting differences in autoantibody activity directed against epitopes in a patient group compared to controls must be resolved against a large background of autoimmune noise. Nevertheless, banding patterns can be quantified and statistically analyzed, permitting detection of minor changes in the antigenic composition of autoantibody repertoires (Nobrega et al., 1993). A multivariate analysis of discriminance can be used to statistically differentiate between the mean banding patterns of groups, as well as to measure the extent to which multiple dependent variables, i.e. Ab activity against specific epitopes or individual Ab repertoires, contribute to differences between groups Ferreira et al., 1997, Grus and Augustin, 1998, Haury et al., 1994, Nobrega et al., 1993, Sharshar et al., 1998, Zimmermann et al., 1995. Furthermore, a discriminant analysis provides the ability to screen simultaneously for a wide spectrum of autoantigens.
In this study, we applied a multivariate statistical method shown to be effective in describing autoantibody repertoires to both new Western blot data and to results previously reported using a basic analytic method Singer et al., 1998, Singer et al., 1999. Sera from children with TS and controls were exposed to epitopes from human striatum, globus pallidus, muscle, and a neuron-like HTB-10 neuroblastoma cell line. The latter was selected because of its suggested ability to differentiate between normal and abnormal populations, and its high degree of sensitivity and specificity (Laurino et al., 1997). We hypothesized that children with TS would have different patterns of autoantibody activity against striatal regions, since alterations of the symmetry or size of the caudate and putamen have been identified in volumetric MRI studies of patients with tic disorders Singer et al., 1993, Hyde et al., 1995, Moriarty et al., 1997. Our goal was to determine if differences in IgG repertoires against these epitopes exist between patient and control populations and to highlight individual molecular weight (MW) ranges that may be specific determinants of an autoimmune process in TS.
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Subjects
Twenty outpatients with TS attending a referral-based clinic (18 boys, 2 girls; age range, 7–16 years; mean, 10.7 years) were voluntary participants in this hospital-approved study. All fulfilled the diagnostic criteria for TS as defined by the Tourette Syndrome Classification Study Group (1993). The mean age of tic onset was 6.6 years (range, 2–11 years) and 65% had a family history of tics. No subject had a history of rheumatic fever, Sydenham's chorea, or autoimmune disease. All TS subjects
Group characteristics
Homogenates of striatal, muscle, globus pallidus, and HTB-10 antigens showed widely different protein compositions and individually unique patterns of antibody reactivity. Blots for all homogenates displayed complex staining patterns, with antibody activity directed at multiple MW ranges. No range showed measurable antibody activity exclusive to one group (TS or control) for any of the four antigenic tissues. Total serum blots against each tissue showed similar mean numbers of measurable
Discussion
Analysis of the serum IgG repertoires of patients with TS and controls against human striatal, globus pallidus, and muscle extracts and the HTB-10 neuroblastoma cell line show highly conserved reactivities against globus pallidus, muscle and HTB-10. In contrast, Ab reactivities against antigens from human striatum differ widely between TS and control groups. Although we could speculate that this difference is due to a prior infection, changes could be due to multiple other factors, including
Acknowledgements
The authors wish to thank Dr. Pamela Talalay for helpful discussion during the preparation of the manuscript. Our research was supported by grants from the National Institutes of Health (N537706) and the National Tourette's Syndrome Association.
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