Figure 1 KCTD16 antibodies are associated with an underlying tumour

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Figure 1 KCTD16 antibodies are associated with an underlying tumour Figure 1 KCTD16 antibodies are associated with an underlying tumour. (A) Bar diagram depicting percentages of patients ... Figure 1 KCTD16 antibodies are associated with an underlying tumour. (A) Bar diagram depicting percentages of patients with or without an underlying tumour. Patients with KCTD16 antibodies more frequently have an underlying tumour. Fisher exact test, P = 0.001. (B) Scatterplot depicting serum and CSF anti-GABA<sub>B</sub>R titres of patients with or without KCTD16 antibodies; lines indicate median values. GABA<sub>B</sub>R antibody titres in serum do not differ between patients with or without KCTD16 antibodies, whereas antibody titres in CSF are significantly higher in patients with KCT16 antibodies. Mann-Whitney test, P = 0.24 (serum), P = 0.01 (CSF). (C) Bar diagram depicting percentages of patients with a status epilepticus. Status epilepticus tended to occur more frequently in patients with KCTD16 antibodies when compared to patients without KCTD16 antibodies. Fisher exact test, P = 0.045 (P-values between 0.01 and 0.05 should be considered with caution). (D) Scatterplot depicting mRS at disease maximum, lines indicate median values. Maximum disease severity does not differ between patients with or without KCTD16 antibodies. Mann-Whitney test, P = 0.59. (E) Scatterplot depicting minimal mRS after treatment, lines indicate median values. Response to treatment does not differ between patients with or without KCTD16 antibodies. Mann-Whitney test, P = 0.20. (F) Immunohistochemistry of SCLC tissue from Patient 5, stained with haematoxylin and eosin (HE), normal rabbit serum and KCTD16 antibody. The image shows specific KCTD16 expression in tumour cells, which is absent in healthy lung tissue. Staining was performed on sequential slides and images were taken in the same area of the sample. Scale bars = 25 µm. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Brain, awz094, https://doi.org/10.1093/brain/awz094 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2 Kaplan Meier curve of survival comparing tumour and non-tumour patients. Showing the data of patients with (n ... Figure 2 Kaplan Meier curve of survival comparing tumour and non-tumour patients. Showing the data of patients with (n = 21) and without tumours (n = 7). Median survival was 17 months (95% CI 7.80–26.20), not different between patients with tumours (15 months, 95% CI 11.04–18.96), or without (no median number as >50% survived, P = 0.36). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Brain, awz094, https://doi.org/10.1093/brain/awz094 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3 Diagnostic tests for GABAB receptor antibodies Figure 3 Diagnostic tests for GABAB receptor antibodies. (A) Immunohistochemistry of adult rat brain ... Figure 3 Diagnostic tests for GABA<sub>B</sub> receptor antibodies. (A) Immunohistochemistry of adult rat brain stained with patient CSF or control CSF. The patient CSF shows brain-wide neuropil staining, here exemplified by an image of the hippocampus. Scale bars = 500 µm. (B) Immunocytochemistry of living rat hippocampal neurons. Labelling with the patient serum (green) results in a dot-like pattern along the neurites. Scale bars = 10 µm. (C) Live in-house CBA of HEK cells transfected with GABA<sub>B1</sub>-GFP and GABA<sub>B2</sub> (green) and stained with patient serum or control serum (red). The patient serum labels the surface of cells transfected with GABA<sub>B</sub> receptor. Scale bars = 20 µm. (D) Bar diagram representing the percentages of positive and negative tests for the different laboratory techniques that are used for the detection of GABA<sub>B</sub> receptor antibodies. For one patient, CSF was not available to perform CBA, but this sample did test positive for GABA<sub>B</sub> receptor in live CBA (Dalmau lab, Barcelona). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Brain, awz094, https://doi.org/10.1093/brain/awz094 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4 Endpoint titrations with fixed cell-based assay Figure 4 Endpoint titrations with fixed cell-based assay. (A) Titration of serum of an anti-KCTD16-negative patient ... Figure 4 Endpoint titrations with fixed cell-based assay. (A) Titration of serum of an anti-KCTD16-negative patient (red) using a fixed CBA of HEK cells transfected with GABA<sub>B1</sub>-GFP and GABA<sub>B2</sub> (green) with or without co-transfection of KCTD16. Staining of cells co-transfected with KCTD16 can be detected up to a dilution of 1:3200, as opposed to without KCTD16 co-transfection, up to a dilution of 1:800. (B) Serum titres detected with a fixed CBA with or without co-transfection of KCTD16. Higher serum titres are detected with the addition of KCTD16 to the CBA. Median serum titre detected with the GABA<sub>B1/2</sub> assay was 200 (IQR 60–1600, range 0–25 600), and with addition of KCTD16 3200 (IQR 3200–9600, range 0–64 000; P < 0.0001). (C) CSF titres detected with or without co-transfection of KCTD16. Higher CSF titres are detected with the addition of KCTD16 to the CBA. Median CSF titre detected with the GABA<sub>B1/2</sub> assay was 64 (IQR 7–160, range 0–512), and with addition of KCTD16 128 (IQR 48–512, range 4–2048; P = 0.001). (D) Serum and CSF titres detected with a fixed CBA without KCTD16 co-transfection. Patients with an underlying tumour do not have higher titres in serum and CSF than patients without an underlying tumour. Mann-Whitney test, serum P = 0.23, CSF P = 0.41. Symbols in B–D refer to individual patients, which are explained in greater detail in Supplementary Tables 1 and 3–5. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Brain, awz094, https://doi.org/10.1093/brain/awz094 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5 GABAB receptor with and without KCTD16 co-expression Figure 5 GABAB receptor with and without KCTD16 co-expression. Schematic representation of the possible ... Figure 5 GABA<sub>B</sub> receptor with and without KCTD16 co-expression. Schematic representation of the possible effect of the addition of KCTD16 to the CBA. (A) GABA<sub>B1</sub> and GABA<sub>B2</sub> subunits expressed without co-expression of KCTD16. (B) GABA<sub>B1</sub> and GABA<sub>B2</sub> subunits expressed with co-expression of KCTD16 resulting in a conformational change in the GABA<sub>B</sub> receptor that allows for more efficient antibody binding. (C) GABA<sub>B1</sub> and GABA<sub>B2</sub> subunits expressed with co-expression of KCTD16 resulting in clustering of GABA<sub>B</sub> receptors via the unknown scaffold protein ‘X’ resulting in more dense antibody labelling. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Brain, awz094, https://doi.org/10.1093/brain/awz094 The content of this slide may be subject to copyright: please see the slide notes for details.