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Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease characterised by eosinophilic hyaline intranuclear inclusions which are widely observed in neuronal and somatic cells.1 ,2 NIID has been considered to be a heterogeneous disease with highly variable clinical manifestations such as neuropathy, cerebellar ataxia and dementia, which may occur concomitantly in certain cases.1–5 Sporadic and familial cases have been reported, and the onset of disease varies from the infantile stages to late middle age. These factors made the antemortem diagnosis of NIID difficult. However, in 2011, we reported that skin biopsy is a useful antemortem diagnostic tool for familial neuronal intranuclear inclusion disease because it detects intranuclear inclusions in the dermal cells.3 Recently, some autopsies of NIID patients with leukoencephalopathy have been reported.4 In this study, we identified intranuclear inclusions in skin biopsy samples from three sporadic NIID patients who presented with cognitive dysfunction along with notable brain MRIs findings of leukoencephalopathy.
A patient aged in the late sixties with neither significant past medical history nor family history of neurological disease was referred to our hospital with gait disturbance and dementia with symptoms including frequent disorientation over 3 years. A neurological examination revealed no ataxia, sensory disturbances or urinary incontinence. The patient's Mini-Mental State Examination (MMSE) Score was 29. A brain MRI showed moderate cerebral and cerebellar atrophy and high-intensity areas in the cerebral white matter in the T2-weighted and fluid-attenuated inversion recovery (FLAIR) images (figure 1A). The MRI diffusion-weighted imaging (DWI) revealed a high-intensity signal in the corticomedullary junction, and these areas showed isointensity and low intensity on the ADC map (figure 1A). A cerebrospinal fluid (CSF) examination showed no pleocytosis or protein elevation and a normal glucose level. The nerve conduction studies were normal.
The patient aged in seventies consulted a neurologist for dementia. The patient had a history of prostate enlargement, and father was diagnosed with Parkinson's disease. At the age of 65 years, this patient noticed unsteady gait and experienced difficulty remembering people's name. Then, the patient began talking to oneself, complained of transient blindness, and began to eat flowers instead of food. A neurological examination revealed dementia, limb-kinetic apraxia, constructional apraxia, dysarthria, wide-based gait and constipation. A brain MRI showed atrophy in the cerebellum and high-intensity areas in bilateral cerebral white matter on the T2 and FLAIR (figure 1A), a high-intensity signal in the corticomedullary junction on the DWI and isointensity and low intensity on the ADC map (figure 1A). An EEG showed a diffuse slow α background, and high voltage θ–δ waves appeared diffusely.
A patient aged in the late sixties presented with abnormal behaviour, such as absentmindedly standing naked in a room. The patient had a past history of gastric ulcer and diabetes but no family history of neurological diseases. The neurological examination revealed miosis with normal light reflex, decreased tendon reflexes and a positive bilateral Babinski sign. The patient's MMSE score was 29. The results of a brain MRI were identical to Case 1 and Case 2 (figure 1A). A CSF examination showed no pleocytosis but did show a slight protein elevation and a slight glucose elevation. An EEG showed no abnormal activity.
Genetic testing of the FMR1 gene for these three cases revealed no expanded CGG premutation. We performed skin biopsies as part of the differential diagnosis for leukoencephalopathy, with the expectation of NIID.
Skin biopsy samples were collected from the patients and normal volunteers under local anesthaesia. A 3-mm-diameter punch biopsy specimen was obtained at 10 cm above the lateral malleolus. One case of Alzheimer's disease and two cases of Binswanger's disease were collected from the autopsy samples. All samples were fixed in 10% formalin and treated as previously reported.3 We performed an immunohistochemical analysis using a Ventana DISCOVERY system (Roche Diagnostics) with anti-ubiquitin antibody (Z0458; DAKO).3 We performed immunofluorescence staining with anti-ubiquitin (Z0458), anti-SUMO1 (sc-5308; Santa Cruz Biotechnology) and anti-p62 (sc-28359; Santa Cruz Biotechnology) as primary antibodies, as previously reported.3 The nuclei were stained with 4′,6-diamidino-2-phenylindole, dilactate (DAPI). The samples for electron microscopy were fixed with glutaraldehyde in cacodylate buffer and embedded in epoxy resin.3
Skin biopsy findings
In the anti-ubiquitin stained sections using the DAB technique (figure 1B) and immunofluorescence technique (figure 1B,C), intranuclear inclusions were identified in the adipocytes, fibroblasts and sweat gland cells of the three patients and no intranuclear inclusion was observed in the normal control or Alzheimer's disease and Binswanger's disease groups (figure 1B,C). The intranuclear inclusions in the dermal cells of these patients also showed anti-SUMO1 and anti-p62 immunoreactivity (figure 1B). Electron microscopy revealed that the intranuclear inclusions of the three patients were composed of filaments and showed no limiting membranes (figure 1D). These results were similar to the results previously reported for NIID neuronal cells.1–3
We reported here sporadic cases of leukoencephalopathy with NIID who presented with cognitive dysfunction. The features of intranuclear inclusions of these cases were identical to those of the familial NIID cases with neuropathy3 ,5 and other reported NIID cases.1 ,2 Our results suggest that sporadic and familial NIID cases could be diagnosed using skin biopsies. Moreover, this technique appears to be useful to diagnose NIID with neuropathy and with leukoencephalopathy. The high intensity of the corticomedullary junction in the DWI is a highly characteristic finding in these cases. An autopsied case with similar MRI findings showed abundant intranuclear inclusions in the cerebral cortex,4 implying that these inclusions may play a pathogenic role. We predict that this MRI observation may also be a new diagnostic clue for NIID and prerequisite for skin biopsy. Given the heterogeneity of the disease, more cases should be further examined to elucidate the pathogenesis and establish the standard diagnostic procedure.
Contributors JS contributed this study by design and conceptualisation, analysis of data and drafting the manuscript. NK contributed this study by acquisition of data and drafting the manuscript. ES contributed this study by acquisition of data and drafting the manuscript. MI contributed this study by acquisition of data and drafting the manuscript. RN contributed this study by analysis of data and drafting the manuscript. HK contributed this study by analysis of data and drafting the manuscript. YI contributed this study by conceptualisation, analysis of data and drafting the manuscript. MY contributed this study by analysis of data and revising the manuscript. TT contributed this study by acquisition of data, analysis of data and drafting the manuscript. SC contributed this study by acquisition of data, analysis of data and drafting the manuscript. MK contributed this study by conceptualisation and revising the manuscript. FT contributed this study by conceptualisation and revising the manuscript. GS contributed this study by design and conceptualisation and revising the manuscript. All the above-mentioned members approved the final version of this paper to be published.
Funding This study was sponsored by a global COE grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a grant from the Ministry of Health, Welfare and Labor of Japan.
Competing interests None.
Patient consent Obtained.
Ethics approval The study was approved by the Institutional Review Board of the Nagoya University School of Medicine.
Provenance and peer review Not commissioned; externally peer reviewed.