Two new cases of anti-Ca (anti-ARHGAP26/GRAF) autoantibody-associated cerebellar ataxia
© Jarius et al.; licensee BioMed Central Ltd. 2013
Received: 27 June 2012
Accepted: 29 November 2012
Published: 15 January 2013
Recently, we discovered a novel serum and cerebrospinal fluid (CSF) autoantibody (anti-Ca) to Purkinje cells in a patient with autoimmune cerebellar ataxia (ACA) and identified the RhoGTPase-activating protein 26 (ARHGAP26; alternative designations include GTPase regulator associated with focal adhesion kinase pp125, GRAF, and oligophrenin-1-like protein, OPHN1L) as the target antigen. Here, we report on two new cases of ARHGAP26 autoantibody-positive ACA that were first diagnosed after publication of the index case study. While the index patient developed ACA following an episode of respiratory infection with still no evidence for malignancy 52 months after onset, neurological symptoms heralded ovarian cancer in one of the patients described here. Our finding of anti-Ca/anti-ARHGAP26 antibodies in two additional patients supports a role of autoimmunity against ARHGAP26 in the pathogenesis of ACA. Moreover, the finding of ovarian cancer in one of our patients suggests that anti-Ca/anti-ARHGAP26-positive ACA might be of paraneoplastic aetiology in some cases. In conclusion, testing for anti-Ca/anti-ARHGAP26 should be included in the diagnostic work-up of patients with ACA, and an underlying tumour should be considered in patients presenting with anti-Ca/ARHGAP26 antibody-positive ACA.
KeywordsAutoimmune cerebellar ataxia Purkinje cells Autoimmunity Autoantibodies RhoGTPase-activating protein 26 (ARHGAP26) GTPase regulator associated with focal adhesion kinase pp125 (GRAF) Oligophrenin-1-like protein Paraneoplastic Ovarian cancer
We recently described a novel serum and cerebrospinal fluid (CSF) autoantibody in a patient with subacute autoimmune cerebellar ataxia (ACA) . In addition, we demonstrated that this antibody (termed anti-Ca), which selectively binds to Purkinje cells when incubated with primate or murine cerebellum tissue sections, targets the RhoGTPase-activating protein 26 (ARHGAP26; alternative designations include GTPase regulator associated with focal adhesion kinase pp125, GRAF, and oligophrenin-1-like protein, OPHN1L).
Here we report on two new cases of ACA with anti-Ca/anti-ARHGAP26 antibodies that were diagnosed since our first publication on this novel serum reactivity. While the index patient had developed ACA following an episode of respiratory infection with still no evidence for cancer 52 months after onset, ACA heralded carcinoma in one of the patients described here, suggesting that anti-Ca/anti-ARHGAP26 is a potential marker of paraneoplastic ACA.
A 38-year-old Iranian man presented with a nine-month history of cerebellar ataxia and dysarthria, weight loss, and vomiting. Brain MRI showed cerebellar atrophy. CSF analysis showed CSF-restricted oligoclonal bands and 5 cells/μl. No further clinical data were available from this patient retrospectively.
IHC was performed on cryosections of adult rhesus monkey, rat, and mouse cerebellum tissue (Euroimmun, Luebeck, Germany) as previously described . Briefly, sections were pretreated with formalin and 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in PBS, blocked with 10% goat serum and then incubated with patient serum for 1 h. Binding of human immunoglobulin (Ig)G, IgA, and IgM to CNS tissue was detected by using polyclonal goat anti-human IgG antibodies conjugated to fluorescein isothiocyanate (FITC) (Euroimmun) or Alexa Fluor™ (AF) 568 (Invitrogen, Karlsruhe, Germany), and polyclonal goat anti-human IgM and anti-human IgA antibodies conjugated to FITC (Euroimmun), respectively. Sections were then mounted using glycerol standard immunofluorescence mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (1:1000) (Euroimmun) or ProLong Gold antifade reagent (Invitrogen). Slides were analyzed on a Nikon 90i upright fluorescence microscope and a Nikon A1 confocal microscope (Nikon Imaging Center, University of Heidelberg, Heidelberg, Germany).
Immunoglobulin G (IgG) subclass analysis
To evaluate IgG subclasses, serum and CSF samples were tested by IHC on mouse cerebellum sections as described above, with the following modifications: sections were blocked with 10% donkey serum; nonconjugated sheep anti-human IgG antibodies specific for IgG subclasses 1 to 4 (Binding site, Germany) were substituted for the FITC-labeled goat anti-human IgG antibody; and AF568-labeled donkey anti-sheep IgG (Invitrogen; absorbed against human IgG) was used to detect the subclass-specific antibodies.
To confirm ARHGAP26 specificity, sera were preadsorbed overnight with human full-length ARHGAP26 (Biozol, Eching, Germany) and supernatants were tested by IHC as described above.
Dot blot assay
Protran BA79 nitrocellulose membranes (0.1 μm) (Whatman, Fisher Scientific, Schwerte, Germany) were spotted with a 0.14 μg/μl solution of human full-length ARHGAP26 (10 μl/spot; Biozol). After drying, membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 1 h at room temperature (RT), washed three times in TBS with 0.05% Tween (TBS-T), and then incubated with a 1:20 dilution of the patient's serum in 0.1% BSA/TBS-T for 1 h at RT. A donkey anti-human IgG antibody labeled with IRdye 700DX (Rockland, Gilbertsville, PA, USA) was used to detect bound IgG. Stripes were finally washed in TBS and analyzed using an Odyssey™ fluorescence scanner (Licor, Lincoln, NE, USA) and Odyssey™ 2.0.40 application software (Licor).
Testing for coexisting antibodies
All samples were tested for anti-Hu, -Yo, -Ri, -CV2/CMRP5, -Ma/Ta, -Tr, -amphiphysin, -aquaporin-4, -GAD, -MAG, and -myelin antibodies by IHC on mouse, rat, and monkey brain and peripheral nerve tissue sections (Euroimmun, Luebeck, Germany), for anti-Hu, -Yo, -Ri, -CV2/CMRP5, -Ma/Ta, and -amphiphysin antibodies by using a commercially available line blot assay (Euroimmun, Germany), and for anti-NMDA-type glutamate receptor, anti-AMPA-type glutamate receptor, anti-GABA-B receptor, anti-CASPR2, anti-LGI1, anti-AQP4, and anti-glycine receptor antibodies using a panel of recombinant cell-based assays (Euroimmun, Germany).
Recently, we reported on a newly discovered serum and CSF autoantibody in a patient with ACA . This new antibody bound selectively to Purkinje cell somata, dendrites, and axons on primate and murine cerebellum tissue sections, and was shown to target ARHGAP26. Here, we report on two newly diagnosed cases of anti-Ca/anti-ARHGAP26-positive ACA.
Occurrence of ARHGAP26 antibody-positive ACA led to the diagnosis of ovarian carcinoma in one of the patients reported here, suggesting a possible paraneoplastic aetiology of the condition. Paraneoplastic neurological disorders count among the most common causes of antibody-associated ACA [2, 3]. It is of note in this context that ARHGAP26 has been shown to be expressed in a subset of ovarian cancer tissues, partly at high levels, while it is absent or present only at low levels in normal ovarian tissue ; however, no tumour tissue from patient 1 was available for analysis in this study.
Antibodies previously demonstrated in patients with paraneoplastic ACA included anti-Hu , anti-Yo , anti-CV2/CRMP5 [7, 8], anti-Tr [9, 10], anti-Zic4 , anti-protein kinase C gamma (PKCγ) , anti-mGluR1 [13, 14], anti-PCA2 , anti-ANNA3 , or voltage-gated calcium channels (VGCC) . None of these antibodies was detected in the patient reported here.
Histologically, a diagnosis of undifferentiated carcinoma was made. However, as a caveat, elevated serum levels of neuron-specific enolase (NSE), a marker of neuroendocrine tumours, were detected. This is of potential interest since tumours of neuroendocrine differentiation such as small-cell lung cancer and neuroblastoma have previously been implicated in a wide range of paraneoplastic neurological disorders, including ACA [2, 18]. As no secondary carcinoma of neuroendocrine differentiation has been found in repeated follow-up examinations, we cannot exclude that the primary tumour contained neuroendocrine components that went unrecognized. Notably, ARHGAP26 has been found to be upregulated in neuroendocrine tumours . Alternatively, the elevated NSE serum levels might be of neuronal origin, reflecting the marked neuronal loss as detected on MRI.
In the second case reported here, the tumour status is unknown as the patient is lost to follow-up; however, the development of ACA was reportedly associated with unusual weight loss in this patient.
Now that it is clear that anti-ARHGAP26/GRAF is present in more patients with ACA, studies on the immunopathological impact of this new serum reactivity are warranted. So far, it is unknown whether the antibody itself causes neurological damage (as has been shown for some of the novel anti-CNS autoantibodies described over the past of couple of years ) or whether the antibody is merely a disease marker of ACA while the actual damage is T cell-mediated (as it is thought to be the case with the classical onco-neuronal antibodies). Of note, as in the index case, anti-Ca/anti-ARHGAP26 belonged to the complement-activating IgG1 subclass in the two new cases reported here, confirming that these new antibodies may possibly act on PCs via complement-dependent mechanisms. In this context, it is of note that patient 1 was, in addition to IgG, positive for IgM antibodies to ARHGAP26. IgM antibodies are generally known to be more potent activators of complement than IgG. Autoantibodies of the IgM class have been reported also in other autoimmune diseases of the CNS . By contrast, patient 2 as well as well as the index patient  were negative for ARHGAP26-IgM.
Our finding of high-titre anti-Ca/anti-ARHGAP26 antibodies in two additional patients with ACA strongly supports a role for autoimmunity against ARHGAP26 in the pathogenesis of this rare condition and proves that the index patient was not a singular case. Moreover, the finding of ovarian cancer in one of our patients suggests that anti-Ca/anti-ARHGAP26-positive ACA might be of paraneoplastic aetiology in some cases. In conclusion, testing for anti-Ca/anti-ARHGAP26 should be included in the diagnostic work-up of patients with ACA; while more cases have to be evaluated before a strict recommendation can be made as to whether broad tumour screening is generally required in patients with anti-Ca/ARHGAP26 antibody-positive ACA, non-harmful screening procedures such as ultrasound examination for ovarian cancer and, possibly, tumour marker testing seem warranted.
Senior authors: Brigitte Wildemann and Klaus-Peter Wandinger.
We are thankful to Annemarie Eschlbeck and Brigitte Fritz as well as to the Nikon Imaging Center at the University of Heidelberg for excellent technical assistance.
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