Contactin-associated protein-2 antibodies in non-paraneoplastic cerebellar ataxia

Abstract

Background: Relatively few studies have searched for potentially pathogenic antibodies in non-paraneoplastic patients with cerebellar ataxia.

Methods and results: We first screened sera from 52 idiopathic ataxia patients for binding of serum IgG antibodies to cerebellar neurons. One strong-binding serum was selected for immunoprecipitation and mass spectrometry, which resulted in the identification of contactin-associated protein 2 (CASPR2) as a major antigen. CASPR2 antibodies were then found by a cell-based assay in 9/88 (10%) ataxia patients, compared to 3/144 (2%) multiple sclerosis or dementia controls (p=0.011). CASPR2 is strongly expressed in the cerebellum, only partly in association with voltage-gated potassium channels.

Conclusions: Prospective studies are now needed to see whether identification of CASPR2 antibodies has relevance for the diagnosis and treatment of idiopathic cerebellar ataxia.

Figure 1

The serum bound strongly, in a punctuate manner, to the axons of cerebellar granule neurons (CGNs) in the molecular layer of unpermeabilised organotypic cerebellar slices (A) and unpermeabilised dissociated CGN cultures (B). GCL, granule cell layer; ML, molecular layer. (C) Gel electrophoresis of immunoprecipitates after incubation of serum with unpermeabilised CGNs followed by solubilisation. The asterisks indicate heavy and light IgG chains, respectively. All precipitated proteins were excised and analysed by mass spectrometry. CASPR2, corresponding to the 180-kDa protein band selectively precipitated by the patient's IgG (arrow), was the only significant membrane protein identified by mass spectrometry (MudPIT scores >41). (D) The index patient serum IgG (red) bound strongly to the CASPR2-EGFP-transfected (green) HEK293T cells (score 4) but the control serum did not bind (score 0). (E) Binding in the cell-based assay was determined at 1:100 serum dilution and scored visually from 0 (no binding) to 4 (very strong binding). Sera scoring 1 or above were considered positive, as in our previous publications, and only if scoring 1 or above in two independent, coded assays. Positive binding to CASPR2 (score 1–4) was detected in nine of 88 (10%) patient sera compared with three of 144 control samples (Fisher's exact test, p=0.011). The horizontal line represents the cut-off above which the results are considered positive. To confirm some of the lower values, available sera were tested to endpoint dilution. Two MS sera scoring 1 and 2 titrated to 1:100 and 1:200; two ataxia sera scoring 1 and 1.5 titrated to 1:200 and 1:400.