Skin biopsy is useful for the antemortem diagnosis of neuronal intranuclear inclusion disease

RESULTS

We performed more than 30 skin biopsies with no adverse reaction or accident. H&E-stained sections from patients with NIID demonstrated eosinophilic intranuclear inclusions in adipocytes, fibroblasts, and sweat gland cells in the dermis (figure 1, A–C). The nuclei of these cells were strongly stained basophilic, which made it difficult to observe inclusions in such small, dark-stained nuclei (figure 1, A–C). In anti-ubiquitin-stained sections using the DAB technique (figure 1, D–F), intranuclear inclusions were identified easily in all 7 NIID samples. However, erythrocytes and some secreted materials from the sweat glands were strongly stained with the anti-ubiquitin antibody, which made it difficult to distinguish these materials from intranuclear inclusions.

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Figure 1 Histopathologic features of neuronal intranuclear inclusion disease (NIID) cells

(A–C) Hematoxylin & eosin (H&E) stain of adipocytes (A), fibroblasts (B), and sweat gland cells (C). (D–F) Immunostained samples of adipocytes (D), fibroblasts (E), and sweat gland cells (F) with anti-ubiquitin antibody using the DAB technique. (G–L) Electron microscopic images of adipocytes (G, J), fibroblasts (H, K), and sweat gland cells (I, L). (G–I) Lower magnification view of intranuclear inclusion (arrow). (J–L) Higher magnification view of each intranuclear inclusion. (M–O) Immunostaining with anti-SUMO1 antibody using the DAB technique of adipocytes (M), fibroblasts (N), and sweat gland cells (O). (P–T) Histopathologic features of intranuclear inclusion of neuronal cells of patients with NIID²: H&E stain of sympathetic ganglion neuron (P); immunostaining with anti-ubiquitin antibody of dorsal root ganglion neuron (Q); electron microscopic images of astrocyte of anterior horn in lower magnification (R) and higher magnification (S); and immunostaining with anti-SUMO1 antibody of sympathetic ganglion neuron (T). (A–F, M–Q, T) Scale bar = 10 μm. (G–I, R) Scale bar = 2.0 μm. (J–L, S) Scale bar = 500 nm.

By examination using electron microscopy, intranuclear inclusions in adipocytes, fibroblasts, and sweat gland cells showed common features (figure 1, G–L). These inclusions were composed of filamentous material and showed no limiting membrane. The nuclei of adipocytes were observed as entirely electron dense, but we were able to identify inclusions in the nuclei of adipocytes (figure 1, G and J). The nuclei of fibroblasts were less electron dense than those of adipocytes, and inclusions were recognized easily as electron-dense spherical bodies and filaments arranged in turbinate fashion (figure 1, H and K). In the sweat gland cells, intranuclear inclusions were observed as electron light material in the nucleus (figure 1, I and L).

We also examined anti-SUMO1 antibody immunoreactivity. SUMO1 is a small ubiquitin-like modifier protein that covalently conjugates to various intracellular target proteins to alter their cellular distribution, function, and metabolism. Neuronal intranuclear inclusions of patients with NIID are immunoreactive for SUMO1.⁹ Intranuclear inclusions in the dermal cells showed anti-SUMO1 immunoreactivity (figure 1, M–O), similar to results previously reported for NIID neuronal cells.^1,9

These features of inclusions are identical to those of neuronal inclusions in patients with NIID (figure 1, P–T).²

Frequency of intranuclear inclusions.

We investigated the frequency of intranuclear inclusions in adipocytes, fibroblasts, and sweat gland cells using anti-ubiquitin antibody and DAPI for the purpose of distinguishing intranuclear inclusions from other ubiquitin-positive materials. Intranuclear inclusions were recognized readily as ubiquitin-positive inclusions within DAPI-positive nuclei under merge view (figure 2A). The frequency of intranuclear inclusion–positive adipocytes in NIID skin samples was approximately 10%, which represented the highest frequency among the 3 cell types (table).

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Figure 2 Immunofluorescence examination of skin samples from patients with neuronal intranuclear inclusion disease (NIID) and other neurodegenerative diseases

(A) Double immunofluorescence staining with anti-ubiquitin antibody and 4′,6-diamidino-2-phenyindole di-lactate (DAPI) in NIID skin samples from family I.² Intranuclear inclusions were stained with anti-ubiquitin antibody (green) and these inclusions are included in the DAPI-positive nuclei in the merged view. Scale bar = 10 μm. (B–M) Double fluorescence staining for adipocytes in dermis with anti-ubiquitin antibody and DAPI in NIID family II² (B), normal control (C), Charcot-Marie-Tooth disease (D), familial amyloid polyneuropathy (E), Huntington disease (F), spinocerebellar ataxia 3 (G), dentatorubral pallidoluysian atrophy (H), spinal and bulbar muscular atrophy (I), amyotrophic lateral sclerosis (J), Parkinson disease (K), multiple system atrophy (L), and progressive supranuclear palsy (M). Scale bars = 10 μm.

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Table

Frequency of intranuclear inclusions in adipocytes, fibroblasts, and sweat gland cells in patients with NIID^a