Evaluation of a qualified MDCK cell line for virus isolation to develop cell-based influenza vaccine viruses with appropriate antigenicity.

We established a qualified Madin-Darby canine kidney cell line (qMDCK-Cs) and investigated its suitability for source virus isolation to develop cell-based seasonal influenza vaccine viruses using vaccine manufacturer cells (Manuf-Cs). When inoculated with 81 influenza-positive clinical specimens, the initial virus isolation efficiency of qMDCK-Cs was exceeded 70%. Among the qMDCK-C isolates, 100% of the A/H1N1pdm09, B/Victoria and B/Yamagata strains and >70% of the A/H3N2 strains showed antigenicity equivalent to that of the contemporary vaccine or relevant viruses in haemagglutination inhibition (HI) or virus neutralization (VN) tests using ferret antisera. These qMDCK-C isolates were propagated in Manuf-Cs (MDCK and Vero cells) (Manuf-C viruses) to develop vaccine viruses. In reciprocal antigenicity tests, ferret antisera raised against corresponding reference viruses and Manuf-C viruses recognized 29 of 31 Manuf-C viruses and corresponding reference viruses, respectively at HI or VN titres more than half of the homologous virus titres, which is the antigenicity criterion for cell culture seasonal influenza vaccine viruses specified by the World Health Organization. Furthermore, ferret antisera against these Manuf-C viruses recognized ≥95% of the viruses circulating during the relevant influenza season with HI or VN titres greater than one-quarter of the homologous virus titres. No cell line-specific amino acid substitutions were observed in the resulting viruses. However, polymorphisms at positions 158/160 of H3HA, 148/151 of N2NA and 197/199 of B/Victoria HA were occasionally detected in the qMDCK-C and Manuf-C viruses but barely affected the viral antigenicity. These results indicated that qMDCK-Cs are suitable for isolating influenza viruses that can serve as a source of antigenically appropriate vaccine viruses. The use of the qMDCK-C isolates will eliminates the need for clinical sample collection, virus isolation, and antigenicity analysis every season, and is expected to contribute to the promotion of vaccine virus development using manufacturer cells.

Retrospective review of bladder biopsy instead of skin biopsy provided clue for the diagnosis of neuronal intranuclear inclusion disease.

The purpose of this study is to present a case of a patient with a negative skin biopsy who was diagnosed with neuronal intranuclear inclusion disease (NIID) through a retrospective review of a bladder biopsy specimen obtained 10 years ago. The patient initially presented with encephalitis-like symptoms, including fever, headache, and abnormal mental behavior. However, the DWI hyperintensity in the corticomedullary junction indicated the possibility of NIID. Due to the negative results of the skin biopsy, we initially refrained from hastily diagnosing the patient with NIID. However, 6 months later, the patient was readmitted to the hospital due to similar symptoms, and a follow-up MRI showed significant enlargement of the lesions at the corticomedullary junction. This time we identified intranuclear inclusions in her tissue specimens from bladder surgery. Subsequently, genetic testing was performed, leading to the diagnosis of NIID in the patient. Our case report indicates that detecting intranuclear inclusions from previous surgical specimens, rather than relying solely on skin biopsy, could significantly enhance diagnostic methods for NIID.

Nerve conduction features may serve as a diagnostic clue for neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease is a neurodegenerative disorder with a wide phenotypic spectrum, including peripheral neuropathy. This study aims to characterize the nerve conduction features and proposes an electrophysiological criterion to assist the diagnosis of neuronal intranuclear inclusion disease. In this study, nerve conduction studies were performed in 50 genetically confirmed neuronal intranuclear inclusion disease patients, 200 age- and sex-matched healthy controls and 40 patients with genetically unsolved leukoencephalopathy. Abnormal electrophysiological parameters were defined as mean values plus or minus two standardized deviations of the healthy controls or failure to evoke a response on the examined nerves. Compared to controls, neuronal intranuclear inclusion disease patients had significantly slower motor and sensory nerve conduction velocities, as well as lower amplitudes of compound motor action potentials and sensory nerve action potentials in all tested nerves (P < 0.05). Forty-eight of the 50 neuronal intranuclear inclusion disease patients (96%) had at least one abnormal electrophysiological parameter, with slowing of motor nerve conduction velocities being the most prevalent characteristic. The motor nerve conduction velocities of median, ulnar, peroneal and tibial nerves were 44.2 ± 5.5, 45.3 ± 6.1, 37.3 ± 5.3 and 35.6 ± 5.1 m/s, respectively, which were 12.4-13.6 m/s slower than those of the controls. The electrophysiological features were similar between neuronal intranuclear inclusion disease patients manifesting with CNS symptoms and those with PNS-predominant presentations. Thirteen of the 14 patients (93%) who underwent nerve conduction study within the first year of symptom onset exhibited abnormal findings, indicating that clinical or subclinical peripheral neuropathy is an early disease marker of neuronal intranuclear inclusion disease. We then assessed the feasibility of using motor nerve conduction velocity as a diagnostic tool of neuronal intranuclear inclusion disease and evaluated the diagnostic performance of various combinations of nerve conduction parameters using receiver operating characteristic curve analysis. The criterion of having at least two nerves with motor nerve conduction velocity ranging from 35 to 50 m/s in median/ulnar nerves and 30-40 m/s in tibial/peroneal nerves demonstrated high sensitivity (90%) and specificity (99%), with an area under the curve of 0.95, to distinguish neuronal intranuclear inclusion disease patients from healthy controls. The criterion's diagnostic performance was validated on an independent cohort of 56 literature reported neuronal intranuclear inclusion disease cases (area under the curve = 0.93, sensitivity = 87.5%, specificity = 99.0%), and in distinguishing neuronal intranuclear inclusion disease from genetically unresolved leukoencephalopathy cases (sensitivity = 90.0%, specificity = 80.0%). In conclusion, mildly to moderately decreased motor nerve conduction velocity in multiple nerves is a significant electrophysiological hallmark assisting the diagnosis of neuronal intranuclear inclusion disease, regardless of CNS- or PNS-predominant manifestations.

Ultra-high-field 7-Tesla magnetic resonance imaging in fragile X tremor/ataxia syndrome (FXTAS).

Fragile X tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder characterized by premutation expansion of fragile X mental retardation 1 (FMR1) gene. It is a common single-gene cause of tremor, ataxia, and cognitive decline in adults. FXTAS affects the central, peripheral and autonomic nervous systems, leading to a range of neurological symptoms from dementia to dysautonomia. A characteristic imaging feature of FXTAS is symmetric T2 hyperintensity in the deep white matter of the cerebellar hemispheres and middle cerebral peduncle. However, recent studies have reported additional findings on diffusion weighted images (DWI), such as a symmetric high-intensity band-like signal at the cerebral corticomedullary junction. These findings, along with the characteristic cerebellar signal alterations, overlap with imaging findings seen in adult-onset neuronal intranuclear inclusion disease (NIID). Importantly, recent pathology studies have shown that both FXTAS and NIID can manifest intranuclear inclusion bodies, posing a diagnostic challenge and potential for misdiagnosis. We describe a 58-year-old man with FXTAS who received an erroneous diagnosis based on imaging and histopathology results. We emphasize the potential pitfalls in distinguishing NIID from FXTAS and stress the importance of genetic analysis in all cases with suspected NIID and FXTAS for confirmation. Additionally, we present the 7T MRI brain findings of FXTAS.

Case report: Neuronal intranuclear inclusion disease initially mimicking reversible cerebral vasoconstriction syndrome: serial neuroimaging findings during an 11-year follow-up.

Neuronal intranuclear inclusion disease (NIID) is a rare, progressive neurodegenerative disorder known for its diverse clinical manifestations. Although episodic neurogenic events can be associated with NIID, no reported cases have demonstrated concurrent clinical features or MRI findings resembling reversible cerebral vasoconstriction syndrome (RCVS). Here, we present the inaugural case of an adult-onset NIID patient who initially displayed symptoms reminiscent of RCVS. The 59-year-old male patient's initial presentation included a thunderclap headache, right visual field deficit, and confusion. Although his brain MRI appeared normal, MR angiography unveiled left posterior cerebral artery occlusion, subsequently followed by recanalization, culminating in an RCVS diagnosis. Over an 11-year period, the patient encountered 10 additional episodes, each escalating in duration and intensity, accompanied by seizures. Simultaneously, cognitive impairment progressed. Genetic testing for NIID revealed an abnormal expansion of GGC repeats in NOTCH2NLC, with a count of 115 (normal range, <60), and this patient was diagnosed with NIID. Our report highlights that NIID can clinically and radiologically mimic RCVS. Therefore, in the differential diagnosis of RCVS, particularly in cases with atypical features or recurrent episodes, consideration of NIID is warranted. Additionally, the longitudinal neuroimaging findings provided the course of NIID over an 11-year follow-up period.

Skin biopsy and neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disease with variable clinical phenotypes. There is a considerable delay in the definite diagnosis, which primarily depends on postmortem brain pathological examination. Although CGG repeat expansion in the 5'-untranslated region of NOTCH2NLC has been identified as a disease-associated variant, the pathological diagnosis is still required in certain NIID cases. Intranuclear inclusions found in the skin tissue of patients with NIID dramatically increased its early detection rate. Skin biopsy, as a minimally invasive method, has become widely accepted as a routine examination to confirm the pathogenicity of the repeat expansion in patients with suspected NIID. In addition, the shared developmental origin of the skin and nerve system provided a new insight into the pathological changes observed in patients with NIID. In this review, we systematically discuss the role of skin biopsy for NIID diagnosis, the procedure of skin biopsy, and the pathophysiological mechanism of intranuclear inclusion in the skin.

The genetic and clinical spectrum in a cohort of 39 families with complex inherited peripheral neuropathies.

With complicated conditions and a large number of potentially causative genes, the diagnosis of a patient with complex inherited peripheral neuropathies (IPNs) is challenging. To provide an overview of the genetic and clinical features of 39 families with complex IPNs from central south China and to optimize the molecular diagnosis approach to this group of heterogeneous diseases, a total of 39 index patients from unrelated families were enrolled, and detailed clinical data were collected. TTR Sanger sequencing, hereditary spastic paraplegia (HSP) gene panel, and dynamic mutation detection in spinocerebellar ataxia (SCAs) were performed according to the respective additional clinical features. Whole-exome sequencing (WES) was used in patients with negative or unclear results. Dynamic mutation detection in NOTCH2NLC and RCF1 was applied as a supplement to WES. As a result, an overall molecular diagnosis rate of 89.7% was achieved. All 21 patients with predominant autonomic dysfunction and multiple organ system involvement carried pathogenic variants in TTR, among which nine had c.349G > T (p.A97S) hotspot variants. Five out of 7 patients (71.4%) with muscle involvement harbored biallelic pathogenic variants in GNE. Five out of 6 patients (83.3%) with spasticity reached definite genetic causes in SACS, KIF5A, BSCL2, and KIAA0196, respectively. NOTCH2NLC GGC repeat expansions were identified in all three cases accompanied by chronic coughing and in one patient accompanied by cognitive impairment. The pathogenic variants, p.F284S and p.G111R in GNE, and p.K4326E in SACS, were first reported. In conclusion, transthyretin amyloidosis with polyneuropathy (ATTR-PN), GNE myopathy, and neuronal intranuclear inclusion disease (NIID) were the most common genotypes in this cohort of complex IPNs. NOTCH2NLC dynamic mutation testing should be added to the molecular diagnostic workflow. We expanded the genetic and related clinical spectrum of GNE myopathy and ARSACS by reporting novel variants.

Advances in Nucleotide Repeat Expansion Diseases: Transcription Gets in Phase.

Unstable DNA repeat expansions and insertions have been found to cause more than 50 neurodevelopmental, neurodegenerative, and neuromuscular disorders. One of the main hallmarks of repeat expansion diseases is the formation of abnormal RNA or protein aggregates in the neuronal cells of affected individuals. Recent evidence indicates that alterations of the dynamic or material properties of biomolecular condensates assembled by liquid/liquid phase separation are critical for the formation of these aggregates. This is a thermodynamically-driven and reversible local phenomenon that condenses macromolecules into liquid-like compartments responsible for compartmentalizing molecules required for vital cellular processes. Disease-associated repeat expansions modulate the phase separation properties of RNAs and proteins, interfering with the composition and/or the material properties of biomolecular condensates and resulting in the formation of abnormal aggregates. Since several repeat expansions have arisen in genes encoding crucial players in transcription, this raises the hypothesis that wide gene expression dysregulation is common to multiple repeat expansion diseases. This review will cover the impact of these mutations in the formation of aberrant aggregates and how they modify gene transcription.

Current advances in neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a rare but probably underdiagnosed neurodegenerative disorder due to pathogenic GGC expansions in the NOTCH2NLC gene. In this review, we summarize recent developments in the inheritance features, pathogenesis, and histopathologic and radiologic features of NIID that subvert the previous perceptions of NIID. GGC repeat sizes determine the age of onset and clinical phenotypes of NIID patients. Anticipation may be absent in NIID but paternal bias is observed in NIID pedigrees. Eosinophilic intranuclear inclusions in skin tissues once considered pathological hallmarks of NIID can also present in other GGC repeat diseases. Diffusion-weighted imaging (DWI) hyperintensity along the corticomedullary junction once considered the imaging hallmark of NIID can frequently be absent in muscle weakness and parkinsonism phenotype of NIID. Besides, DWI abnormalities can appear years after the onset of predominant symptoms and may even disappear completely with disease progression. Moreover, continuous reports of NOTCH2NLC GGC expansions in patients with other neurodegenerative diseases lead to the proposal of a new concept of NOTCH2NLC-related GGC repeat expansion disorders (NRED). However, by reviewing the previous literature, we point out the limitations of these studies and provide evidence that these patients are actually suffering from neurodegenerative phenotypes of NIID.

GGC repeat expansion in NOTCH2NLC induces dysfunction in ribosome biogenesis and translation.

GGC repeat expansion in the 5' untranslated region (UTR) of NOTCH2NLC is associated with a broad spectrum of neurological disorders, especially neuronal intranuclear inclusion disease (NIID). Studies have found that GGC repeat expansion in NOTCH2NLC induces the formation of polyglycine (polyG)-containing protein, which is involved in the formation of neuronal intranuclear inclusions. However, the mechanism of neurotoxicity induced by NOTCH2NLC GGC repeats is unclear. Here, we used NIID patient-specific induced pluripotent stem cell (iPSC)-derived 3D cerebral organoids (3DCOs) and cellular models to investigate the pathophysiological mechanisms of NOTCH2NLC GGC repeat expansion. IPSC-derived 3DCOs and cellular models showed the deposition of polyG-containing intranuclear inclusions. The NOTCH2NLC GGC repeats could induce the upregulation of autophagic flux, enhance integrated stress response and activate EIF2α phosphorylation. Bulk RNA sequencing for iPSC-derived neurons and single-cell RNA sequencing (scRNA-seq) for iPSC-derived 3DCOs revealed that NOTCH2NLC GGC repeats may be associated with dysfunctions in ribosome biogenesis and translation. Moreover, NOTCH2NLC GGC repeats could induce the NPM1 nucleoplasm translocation, increase nucleolar stress, impair ribosome biogenesis and induce ribosomal RNA sequestration, suggesting dysfunction of membraneless organelles in the NIID cellular model. Dysfunctions in ribosome biogenesis and phosphorylated EIF2α and the resulting increase in the formation of G3BP1-positive stress granules may together lead to whole-cell translational inhibition, which may eventually cause cell death. Interestingly, scRNA-seq revealed that NOTCH2NLC GGC repeats may be associated with a significantly decreased proportion of immature neurons while 3DCOs were developing. Together, our results underscore the value of patient-specific iPSC-derived 3DCOs in investigating the mechanisms of polyG diseases, especially those caused by repeats in human-specific genes.

Subclinical peripheral neuropathy is common in neuronal intranuclear inclusion disease with dominant encephalopathy.

BACKGROUND AND PURPOSE: Neuronal intranuclear inclusion disease (NIID) is associated with CGG repeat expansion in the NOTCH2NLC gene. Although pure or dominant peripheral neuropathy has been described as a subtype of NIID in a few patients, most NIID patients predominantly show involvements of the central nervous system (CNS). It is necessary to further explore whether these patients have subclinical peripheral neuropathy. METHODS: Twenty-eight NIID patients, clinically characterized by CNS-dominant involvements, were recruited from two tertiary hospitals. Standard nerve conduction studies were performed in all patients. Skin and sural nerve biopsies were performed in 28 and 15 patients, respectively. Repeat-primed polymerase chain reaction and amplicon length polymerase chain reaction were used to screen the CGG repeat expansion in NOTCH2NLC. RESULTS: All 28 patients can be diagnosed with NIID based on skin pathological and genetic changes. All patients predominantly showed CNS symptoms mainly characterized by episodic encephalopathy and cognitive impairments, but no clinical symptoms of peripheral neuropathy could be observed initially. Electrophysiological abnormalities were found in 96.4% (27/28) of these patients, indicating that subclinical peripheral neuropathy is common in NIID patients with CNS-dominant type. Electrophysiological and neuropathological studies revealed that demyelinating degeneration was the main pathological pattern in these patients, although mild axonal degeneration was also observed in some patients. No significant association between CGG repeat size and the change of nerve conduction velocity was found in these patients. CONCLUSIONS: This study demonstrated that most patients with CNS-dominant NIID had subclinical peripheral neuropathy. Electrophysiological examination should be the routinely diagnostic workflow for every NIID patient.

Recurrent headache and visual symptoms in a young man: a rare neuronal intranuclear inclusion disease case report.

BACKGROUND: Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease. Patients with NIID may present with heterogeneous clinical symptoms, including episodic encephalopathy, dementia, limb weakness, cerebellar ataxia, and autonomic dysfunction. Among the NIID cases reported in China, patients often have complicated and severe manifestations. Therefore, many clinicians do not consider the disease when the patient presents with relatively minor complaints. CASE PRESENTATION: We present the case of a 39-year-old man showing migraine-aura-like symptoms for the past 3 years. Brain magnetic resonance imaging (MRI) revealed hyperintense signals in the splenium of the corpus callosum and corticomedullary junction on diffusion-weighted imaging (DWI) over time. In addition, brain atrophy that was not concomitant with the patient's age was detected while retrospectively reviewing the patient's imaging results. Genetic analysis and skin biopsy confirmed a diagnosis of NIID. The patient was treated with sibelium, and the symptoms did not recur. DISCUSSION AND CONCLUSIONS: Migraine-aura-like symptoms may be the predominant clinical presentation in young patients with NIID. Persistent high-intensity signals on DWI in the brain and early-onset brain atrophy might be clues for the diagnosis of NIID.

Adult-Onset Neuronal Intranuclear Inclusion Disease with Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-Like (MELAS-like) Episode: A Case Report and Review of Literature.

Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disease with highly heterogeneous manifestations. Curvilinear hyperintensity along the corticomedullary junction on diffusion-weighted images (DWI) is a vital clue for diagnosing NIID. DWI hyperintensity tends to show an anterior-to-posterior propagation pattern as the disease progresses. The rare cases of its disappearance may lead to misdiagnosis. Here, we reported a NIID patient with mitochondrial encephalomyopathy, lactic acidosis and stroke-like (MELAS-like) episode, and reversible DWI hyperintensities. A review of the literature on NIID with MELAS-like episodes was conducted. A 69-year-old woman stated to our clinics for recurrent nausea/vomiting, mixed aphasia, altered mental status, and muscle weakness for 2 weeks. Neurological examination showed impaired mental attention and reaction capacity, miosis, mixed aphasia, decreased muscle strength in limbs, and reduced tendon reflex. Blood tests were unremarkable. The serological examination was positive for antibody against dipeptidyl-peptidase-like protein 6 (DPPX) (1:32). Brain magnetic resonance imaging (MRI) revealed hyperintensities in the left temporal occipitoparietal lobe on DWI and correspondingly elevated lactate peak in the identified restricted diffusion area on magnetic resonance spectroscopy, mimicking the image of MELAS. Skin biopsy and genetic testing confirmed the diagnosis of NIID. Pulse intravenous methylprednisolone and oral prednisolone were administered, ameliorating her condition with improved neuroimages. This case highlights the importance of distinguishing NIID and MELAS, and reversible DWI hyperintensities can be seen in NIID.

Cognitive Dysfunction in Repeat Expansion Diseases: A Review.

With the development of the sequencing technique, more than 40 repeat expansion diseases (REDs) have been identified during the past two decades. Moreover, the clinical features of these diseases show some commonality, and the nervous system, especially the cognitive function was affected in part by these diseases. However, the specific cognitive domains impaired in different diseases were inconsistent. Here, we survey literature on the cognitive consequences of the following disorders presenting cognitive dysfunction and summarizing the pathogenic genes, epidemiology, and different domains affected by these diseases. We found that the cognitive domains affected in neuronal intranuclear inclusion disease (NIID) were widespread including the executive function, memory, information processing speed, attention, visuospatial function, and language. Patients with C9ORF72-frontotemporal dementia (FTD) showed impairment in executive function, memory, language, and visuospatial function. While in Huntington's disease (HD), the executive function, memory, and information processing speed were affected, in the fragile X-associated tremor/ataxia syndrome (FXTAS), executive function, memory, information processing speed, and attention were impaired. Moreover, the spinocerebellar ataxias showed broad damage in almost all the cognitive domains except for the relatively intact language ability. Some other diseases with relatively rare clinical data also indicated cognitive dysfunction, such as myotonic dystrophy type 1 (DM1), progressive myoclonus epilepsy (PME), Friedreich ataxia (FRDA), Huntington disease like-2 (HDL2), and cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS). We drew a cognitive function landscape of the related REDs that might provide an aspect for differential diagnosis through cognitive domains and effective non-specific interventions for these diseases.

Adult-onset autosomal dominant leukodystrophy and neuronal intranuclear inclusion disease: lessons from two new Chinese families.

INTRODUCTION: Adult-onset autosomal dominant leukodystrophy (ADLD) is a rare genetic leukoencephalopathy caused by duplication of the lamin B1 gene (LMNB1) or LMNB1 upstream deletions. Neuronal intranuclear inclusion disease (NIID) is another leukoencephalopathy due to GGC repeat expansion in the 5'-untranslated region of the NOTCH2NLC gene. Here, we report two Chinese ADLD families with neuroimaging and clinical features mimicking NIID. METHODS: We conducted detailed medical history inquiry, neurological examinations, and magnetic resonance imaging in the two families. Candidate gene sequencing and whole exome sequencing (WES) with copy number variation analysis were used to screen the genetic variations. The special points on the clinical and neuroimaging findings in the current families and differential diagnosis of ADLD with NIID are discussed. RESULTS: The two families presented with slowly progressive, multiple central nervous system symptoms, including spastic paraplegia, autonomic dysfunction, ataxia, deep sensory loss, and tremor. Clinical phenotypes were consistent within the family. Transient hypoglycemia and transient dilated pupils indicating autonomic dysfunctions were recorded for the first time in ADLD. Brain MRI showed band-like hyperintensities at the cortico-medullary junction on DWI, typical for NIID. Skin biopsy and genetic sequencing of the NOTCH2NCL gene did not support the diagnosis of NIID. Further whole exome sequencing (WES) identified the duplication mutation spanning the entire LMNB1 gene. CONCLUSIONS: The novel feature of transient hypoglycemia and dilated pupils broadens the spectrum of autonomic dysfunction in ADLD. Clinical manifestations and neuroimaging of ADLD can mimic NIID. Although ADLD is even rarer than NIID, the differential diagnosis of these two diseases should not be confused.

Hornerin deposits in neuronal intranuclear inclusion disease: direct identification of proteins with compositionally biased regions in inclusions.

Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disorder, characterized by the presence of eosinophilic inclusions (NIIs) within nuclei of central and peripheral nervous system cells. This study aims to identify the components of NIIs, which have been difficult to analyze directly due to their insolubility. In order to establish a method to directly identify the components of NIIs, we first analyzed the huntingtin inclusion-rich fraction obtained from the brains of Huntington disease model mice. Although the sequence with expanded polyglutamine could not be identified by liquid-chromatography mass spectrometry, amino acid analysis revealed that glutamine of the huntingtin inclusion-rich fraction increased significantly. This is compatible with the calculated amino acid content of the transgene product. Therefore, we applied this method to analyze the NIIs of diseased human brains, which may have proteins with compositionally biased regions, and identified a serine-rich protein called hornerin. Since the analyzed NII-rich fraction was also serine-rich, we suggested hornerin as a major component of the NIIs. A specific distribution of hornerin in NIID was also investigated by Matrix-assisted laser desorption/ionization imaging mass spectrometry and immunofluorescence. Finally, we confirmed a variant of hornerin by whole-exome sequencing and DNA sequencing. This study suggests that hornerin may be related to the pathological process of this NIID, and the direct analysis of NIIs, especially by amino acid analysis using the NII-rich fractions, would contribute to a deeper understanding of the disease pathogenesis.

Assessing the NOTCH2NLC GGC repeat expansion in Taiwanese patients with hereditary spastic paraplegia.

We investigated 98 Taiwanese patients with molecularly unassigned hereditary spastic paraplegia (HSP) and found none of them had the NOTCH2NLC GGC repeat expansion, which is the cause of neuronal intranuclear inclusion disease (NIID). Our findings suggest that the NOTCH2NLC GGC repeat expansion may not contribute to HSP.