Neuronal and glial intranuclear inclusions in a patient with oculopharyngodistal myopathy associated with noncoding GGC repeat expansions in GIPC1.

Noncoding repeat expansions cause various neuromuscular diseases, including myotonic dystrophies, fragile X tremor/ataxia syndrome, some spinocerebellar ataxias, amyotrophic lateral sclerosis and benign adult familial myoclonic epilepsies. Inspired by the striking similarities in the clinical and neuroimaging findings between neuronal intranuclear inclusion disease (NIID) and fragile X tremor/ataxia syndrome caused by noncoding CGG repeat expansions in FMR1, we directly searched for repeat expansion mutations and identified noncoding CGG repeat expansions in NBPF19 (NOTCH2NLC) as the causative mutations for NIID. Further prompted by the similarities in the clinical and neuroimaging findings with NIID, we identified similar noncoding CGG repeat expansions in two other diseases: oculopharyngeal myopathy with leukoencephalopathy and oculopharyngodistal myopathy, in LOC642361/NUTM2B-AS1 and LRP12, respectively. These findings expand our knowledge of the clinical spectra of diseases caused by expansions of the same repeat motif, and further highlight how directly searching for expanded repeats can help identify mutations underlying diseases.

Neuronal intranuclear inclusion disease (NIID) is a slowly progressive neurodegenerative disease characterized by localized neuronal loss, and the presence of eosinophilic intranuclear inclusions in neurons and glial cells. Biopsy samples of skin, rectal and sural nerve showed hyaline intranuclear inclusions. Reported NIID cases showed familial type according to family history, and three clinical subgroups (infantile, juvenile and adult form) according to onset and disease duration. NIID has been considered as a heterogeneous disease because of the highly variable clinical manifestations including cognitive dysfunction, parkinsonism, cerebellar ataxia, peripheral neuropathy and autonomic dysfunction. Additionally, some NIID cases presented episodes of conscious disturbance, cognitive decline, movement disorder or even fever. Head magnetic resonance imaging of some patients revealed symmetrical leukoencephalopathy in T2 image and fluid attenuated inversion recovery image and high intensity signal in corticomedullary junction in diffusion weighted image. But it is not the only abnormal finding of imaging, and other diseases may also present the similar changing. Other diseases including fragile X-associated tremor-ataxia syndrome, Huntington′s disease and spinocerebellar ataxia should be considered in differential diagnosis. Key words: Neuronal intranuclear inclusion disease; Neurodegenerative disease; Dementia; Peripheral neuropathy; Fragile X-associated tremor-ataxia syndrome

Neuronal intranuclear inclusion disease (NIID) used to be considered as a neurodegenerative disease. Due to the availability of skin biopsy, the diagnostic efficiency of the disease has been greatly improved. Recently, researchers have successfully identified that the GGC repeat expansion in the 5'-untranslated region of the NOTCH2NLC gene is the causative mutation of NIID. Besides the typical phenotype of brain degeneration, peripheral neuropathy, and autonomic disturbance, the gene mutation is also associated with Alzheimer's disease, frontotemporal dementia, Parkinson's disease, multiple system atrophy, essential tremor, adult leukoencephalopathy, and oculopharyngodistal myopathy. However, it still needs more studies to elucidate whether those variable NIID phenotypes can categorize into NOTCH2NLC repeat expansion related disorders. We update the discovery milestone, clinical phenotype, laboratory examinations, as well as new insight into the diagnosis and treatment of NIID. NIID is an unusual degenerative disease that can involve multiple systems, especially involves the nervous system. Originally, it is named after the pathological characteristics with extensive intranuclear eosinophilic inclusions in central and peripheral nervous tissues, as well as in multiple other organs (Sone et al., Brain 139:3170-3186, 2016). In 2019, several research teams from China and Japan have simultaneously identified that the GGC repeat expansion in the 5'-untranslated region (5'UTR) of the NOTCH2NLC gene is the pathogenic mutation of NIID (Ishiura et al., Nat Genet 51:1222-1232, 2019; Deng et al., J Med Genet 56:758-764, 2019; Sone et al., Nat Genet 51:1215-1221, 2019; Sun et al., Brain 143:222-233, 2020; Tian et al., Am J Hum Genet 105:166-176, 2019). Since then, the number of reported NIID cases is rapidly increasing, and the spectrum of NOTCH2NLC repeat expansion related disorders is significantly broadening (Westenberger and Klein, Brain 143:5-8, 2020). However, the NIID associated with GGC repeat expansion of the NOTCH2NLC gene might be account for a part of patients, probably more frequently in the Asian population, because this expansion has not been identified in an European series with postmortem confirmed NIID cases (Chen et al., Ann Clin Transl Neurol 2020). In order to better understand of the disease, we need to revisit the current state of NIID in combination with the findings based on our experiences in recent years and update the concepts about the clinical and pathogenic progression of NIID.

Oculopharyngodistal myopathy (OPDM) is caused by the expansion of CGG repeats in NOTCH2NLC (OPDM_NOTCH2NLC) GIPC1 (OPDM_GIPC1), or LRP12 (OPDM_LRP12). Neuronal intranuclear inclusion disease (NIID) is clinically distinct from OPDM but is also caused by the expansion of CGG repeats in NOTCH2NLC, which may be an indicator of intranuclear inclusion in skin biopsy. We investigated the presence of intranuclear inclusions in skin biopsies from patients with OPDM and muscle diseases with a similar pathology to evaluate whether they will have similar diagnostic findings on skin biopsy. We analysed the frequency of p62-positive intranuclear inclusions in sweat gland cells, adipocytes and fibroblasts in skin biopsy samples from patients with OPDM (OPDM_NOTCH2NLC [n = 2], OPDM_GIPC1 [n = 6] and OPDM_LRP12 [n = 3]), NIID (n = 1), OPMD (n = 1), IBM (n = 4) and GNE myopathy (n = 2). The p62-postive intranuclear inclusions were observed in all three cell types in both patients with OPDM_NOTCH2NLC and a patient with NIID, in at least one cell type in all six patients with OPDM_GIPC1, and all in three cell types in one of the three patients with OPDM_LRP12. These findings were not observed in patients with OPMD, IBM or GNE myopathy. Intranuclear inclusions in skin biopsy samples are not specific to NIID and are found in all three types of genetically confirmed OPDM, suggesting that the underlying mechanism of OPDM may be similar to NIID, regardless of causative genes.

Distribution and frequency of intranuclear inclusions in female CGG KI mice modeling the fragile X premutation

BackgroundBoth fragile X-associated tremor/ataxia syndrome (FXTAS) and late-onset neuronal intranuclear inclusion disease (NIID) show CGG/GGC trinucleotide repeat expansions. Differentiating these diseases are difficult because of the similarity in their clinical and radiological features. It is unclear that skin biopsy can distinguish NIID from FXTAS. We performed a skin biopsy in an FXTAS case with cognitive dysfunction and peripheral neuropathy without tremor, which was initially suspected to be NIID.Case presentationThe patient underwent neurological assessment and examinations, including laboratory tests, electrophysiologic test, imaging, skin biopsy, and genetic test. A brain MRI showed hyperintensity lesions along the corticomedullary junction on diffusion-weighted imaging (DWI) in addition to middle cerebellar peduncle sign (MCP sign). We suspected NIID from the clinical picture and the radiological findings, and performed a skin biopsy. The skin biopsy specimen showed ubiquitin- and p62-positive intranuclear inclusions, suggesting NIID. However, a genetic analysis for NIID using repeat-primed polymerase chain reaction (RP-PCR) revealed no expansion detected in the Notch 2 N-terminal like C (NOTCH2NLC) gene. We then performed genetic analysis for FXTAS using RP-PCR, which revealed a repeat CGG/GGC expansion in the FMRP translational regulator 1 (FMR1) gene. The number of repeats was 83. We finally diagnosed the patient with FXTAS rather than NIID.ConclusionsFor the differential diagnosis of FXTAS and NIID, a skin biopsy alone is insufficient; instead, genetic analysis, is essential. Further investigations in additional cases based on genetic analysis are needed to elucidate the clinical and pathological differences between FXTAS and NIID.

The fragile X premutation is a CGG trinucleotide repeat expansion between 55 and 200 repeats in the 5′-untranslated region of the fragile X mental retardation 1 (FMR1) gene. Human carriers of the premutation allele are at risk of developing the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Characteristic neuropathology associated with FXTAS includes intranuclear inclusions in neurons and astroglia. Previous studies recapitulated these histopathological features in neurons in a knock-in mouse model, but without significant astroglial pathology. To determine the role of astroglia in FXTAS, we generated a transgenic mouse line (Gfa2-CGG99-eGFP) that selectively expresses a 99-CGG repeat expansion linked to an enhanced green fluorescent protein (eGFP) reporter in astroglia throughout the brain, including cerebellar Bergmann glia. Behaviorally these mice displayed impaired motor performance on the ladder-rung test, but paradoxically better performance on the rotarod. Immunocytochemical analysis revealed that CGG99-eGFP co-localized with GFAP and S-100ß, but not with NeuN, Iba1, or MBP, indicating that CGG99-eGFP expression is specific to astroglia. Ubiquitin-positive intranuclear inclusions were found in eGFP-expressing glia throughout the brain. In addition, intracytoplasmic ubiquitin-positive inclusions were found outside the nucleus in distal astrocyte processes. Intriguingly, intranuclear inclusions, in the absence of eGFP mRNA and eGFP fluorescence, were present in neurons of the hypothalamus and neocortex. Furthermore, intranuclear inclusions in both neurons and astrocytes displayed immunofluorescent labeling for the polyglycine peptide FMRpolyG, implicating FMRpolyG in the pathology found in Gfa2-CGG99 mice. Considered together, these results show that Gfa2-CGG99 expression in mice is sufficient to induce key features of FXTAS pathology, including formation of intranuclear inclusions, translation of FMRpolyG, and deficits in motor function.

Amyotrophic lateral sclerosis (ALS) is one of the differential diagnoses of diseases that occur in adulthood and lead to progressive generalized muscle weakness. Neuronal intranuclear inclusion disease (NIID) is a disease in which histopathologically eosinophilic nuclear inclusion bodies are found in various systems. Both familial and sporadic forms of the disease have been reported. Most cases of sporadic NIID are of the dementia type, in which the main symptom is dementia at the first onset. Familial NIID is more diverse, with the main dominant symptoms being muscle weakness (NIID-M), dementia (NIID-D), and parkinsonism (NIID-P). Furthermore, recently, a GGC-repeat expansion in the Notch 2 N-terminal like C (NOTCH2NLC) gene, which produces a toxic polyglycine-containing protein (uN2CpolyG) in patients with NIID, has been associated with the pathogenesis of ALS. These results suggest that sporadic NIIDs may have more diverse forms. To date, no autopsy cases of NIID patients with an ALS phenotype have been reported. Here, we describe the first autopsy case report of a patient with sporadic NIID who had been clinically diagnosed with ALS. A 65-year-old Japanese man with no family history of neuromuscular disease developed progressive muscle atrophy and weakness in all limbs. The patient was diagnosed with ALS (El Escoriral diagnostic criteria: probable ALS, laboratory-supported ALS). He had no cognitive dysfunction or neuropathies suggestive of NIID. He required respiratory assistance 48 months after onset. He died of pneumonia at the age of 79 years. Postmortem examinations revealed neuronal loss in the spinal anterior horns and motor cortex. In these affected regions, eosinophilic, round neuronal intranuclear inclusions were evident, which were immunopositive for ubiquitin, p62, and uN2CpolyG. No Bunina bodies or TDP-43-positive inclusions were observed in the brain or spinal cord. Our findings suggest that a small proportion of patients with NIID can manifest a clinical phenotype of ALS. Although skin biopsy is commonly used for the clinical diagnosis of NIID, it may also be useful to identify cases of NIID masquerading as ALS.

In the case report by Motoki and colleagues, the authors describe a patient with mild positional and intentional tremor, and pathologic confirmation of eosinophilic inclusions in superficial tissues, concerning for neuronal intranuclear inclusion disease (NIID). The patient had undergone renal biopsy 12 years earlier, which also showed eosinophilic intranuclear inclusions that are characteristic of this condition. Of interest, the patient was reportedly neurologically asymptomatic at the time of the renal biopsy, and brain MRI demonstrated a minimal periventricular white matter disease burden, which is etiologically nonspecific. The case highlights the multiorgan involvement of NIID with inclusion deposition that vastly predates neurologic symptom onset. Although the identification of eosinophilic hyaline intranuclear inclusions in fibroblast, adipose, skin, and sweat gland cells has been described in NIID, López-Blanco and colleagues state that similar findings can be found in patients with fragile X-associated tremor/ataxia syndrome (FXTAS). In response, Motoki et al. maintain that testing for FXTAS was unnecessary, given the negative family history, an otherwise normal cerebellar examination, and lack of classic middle cerebellar peduncle hyperintensities on T2-weighted imaging. Recognizing the clinical heterogeneity of FXTAS, López-Blanco et al. defend their comment by citing evidence that absent family history and classic neuroimaging findings are imperfectly sensitive. More importantly would be the implications of passing on the FXTAS mutation to the daughters of the patient. López-Blanco et al. reiterate the need for genetic testing in these uncertain circumstances, along with family counseling, should a mutation be identified. In the case report by Motoki and colleagues, the authors describe a patient with mild positional and intentional tremor, and pathologic confirmation of eosinophilic inclusions in superficial tissues, concerning for neuronal intranuclear inclusion disease (NIID). The patient had undergone renal biopsy 12 years earlier, which also showed eosinophilic intranuclear inclusions that are characteristic of this condition. Of interest, the patient was reportedly neurologically asymptomatic at the time of the renal biopsy, and brain MRI demonstrated a minimal periventricular white matter disease burden, which is etiologically nonspecific. The case highlights the multiorgan involvement of NIID with inclusion deposition that vastly predates neurologic symptom onset.

CSF tau phosphorylated at threonine 181 (p-tau181) is a widely used biomarker for Alzheimer disease (AD) and has recently been regarded to reflect β-amyloid and/or p-tau deposition in the AD brain. Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disease characterized by intranuclear inclusions in neurons, glial cells, and other somatic cells. Symptoms include dementia, neuropathy, and others. CSF biomarkers were not reported. The objective of this study was to investigate whether CSF biomarkers including p-tau181 are altered in patients with NIID. This was a retrospective observational study. CSF concentrations of p-tau181, total tau, amyloid-beta 1-42 (Aβ42), monoamine metabolites homovanillic acid (HVA), and 5-hydroxyindole acetic acid (5-HIAA) were compared between 12 patients with NIID, 120 patients with Alzheimer clinical syndrome biologically confirmed based on CSF biomarker profiles, and patients clinically diagnosed with other neurocognitive disorders (dementia with Lewy bodies [DLB], 24; frontotemporal dementia [FTD], 13; progressive supranuclear palsy [PSP], 21; and corticobasal syndrome [CBS], 13). Amyloid PET using Pittsburgh compound B (PiB) was performed in 6 patients with NIID. The mean age of patients with NIID, AD, DLB, FTD, PSP, and CBS was 71.3, 74.6, 76.8, 70.2, 75.5, and 71.9 years, respectively. CSF p-tau181 was significantly higher in NIID (72.7 ± 24.8 pg/mL) compared with DLB, PSP, and CBS and was comparable between NIID and AD. CSF p-tau181 was above the cutoff value (50.0 pg/mL) in 11 of 12 patients with NIID (91.7%). Within these patients, only 2 patients showed decreased CSF Aβ42, and these patients showed negative or mild local accumulation in PiB PET, respectively. PiB PET scans were negative in the remaining 4 patients tested. The proportion of patients with increased CSF p-tau181 and normal Aβ42 (A-T+) was significantly higher in NIID (75%) compared with DLB, PSP, and CBS (4.2%, 4.8%, and 7.7%, respectively). CSF HVA and 5-HIAA concentrations were significantly higher in patients with NIID compared with disease controls. CSF p-tau181 was increased in patients with NIID without amyloid accumulation. Although the deposition of p-tau has not been reported in NIID brains, the molecular mechanism of tau phosphorylation or secretion of p-tau may be altered in NIID.

Neuronal intranuclear inclusion disease (NIID), also known as neuronal intranuclear hyaline inclusion disease, is a rare neurodegenerative disorder with onset typically in childhood, although adult onset has been described. Both sporadic and familial occurrences have been reported.1,2 Presenting features of NIID are typically widespread, involving central, peripheral, and autonomic nervous systems, leading to the impression of multisystem degeneration.1 These features include parkinsonism, cerebellar ataxia, chorea, dystonia, gaze-evoked horizontal nystagmus, pyramidal tract signs, seizures, mental retardation, dementia, autonomic dysfunction, and peripheral neuropathy. Oculogyric crisis3 and early-onset l-dopa–induced dyskinesias4,5 may be a clue to diagnosis, especially if coexisting gastrointestinal dysfunction is present,2 with pseudoobstruction being relatively common. Clinical features observed in NIID are also typically observed in trinucleotide repeat disorders and therefore NIID should be even more strongly suspected after genetic testing has ruled out trinucleotide repeat disorders. Furthermore, …

<h3>Objective:</h3> To investigate GGC repeat expansion in the 5'untranslated region of <i>NOTCH2NLC</i>, the genetic cause of neuronal intranuclear inclusion disease (NIID), and its contribution to vascular leukoencephalopathy. <h3>Background:</h3> NIID is an autosomal-dominantly inherited neurodegenerative disease with pathological hallmarks of eosinophilic, ubiquitin-, and p62-positive intranuclear inclusions in cells of the nervous systems, skin, and visceral organs. NIID has several clinico-radiological features akin to cerebral small vessel disease (cSVD). Besides, ubiquitin- and p62-positive staining had been detected in the blood vessels of ten autopsied cases with NIID, suggesting a link between NIID and vascular pathology. <h3>Design/Methods:</h3> We screened 197 unrelated patients with vascular leukoencephalopathy and 730 healthy individuals for <i>NOTCH2NLC</i> GGC repeat expansion using repeat-primed PCR, fragment analysis, southern blot analysis, and/or nanopore sequencing with Cas9-mediated enrichment. Skin biopsy with immuno-histochemistry staining and electric microscopic imaging was performed. For patients with vascular leukoencephalopathy, the clinical and neuroimaging characteristics were compared between individuals with and without <i>NOTCH2NLC</i> GGC repeat expansion. <h3>Results:</h3> Six of the 197 patients with vascular leukoencephalopathy carried <i>NOTCH2NLC</i> GGC repeat expansion, while none of the 730 controls harbored the expanded GGC repeats (3.0% vs. 0%, <i>p</i> = 0.00009). Skin biopsy revealed ubiquitin-, and p62-positive intranuclear inclusions in the cells of sweat gland and capillary, providing a pathologic evidence for the involvement of small vessels in NIID. Comparing vascular leukoencephalopathy patients with and without the GGC expansion, there was no difference in the clinical manifestations and severity of cSVD imaging markers, including white matter hyperintensities, enlarged perivascular spaces, lacunes, and cerebral microbleeds. Characteristic imaging features of NIID were detected in five of the six patients carrying GGC expansion and manifesting with vascular leukoencephalopathy. <h3>Conclusions:</h3> The <i>NOTCH2NLC</i> GGC repeat expansion accounts for 3% of the patients with genetically undetermined vascular leukoencephalopathy in the Taiwanese cohort, suggesting that NIID is an under-diagnosed and important cause of cSVD. <b>Disclosure:</b> Dr. Liao has nothing to disclose. Prof. Chung has nothing to disclose. Dr. Lee has nothing to disclose.

Recently, inspired by the similar clinical and pathological features shared with fragile X-associated tremor/ataxia syndrome (FXTAS), abnormal expansion of CGG repeats in the 5’ untranslated region has been found in neuronal intranuclear inclusion disease (NIID), oculopharyngeal myopathy with leukoencephalopathy (OPML), and oculopharyngodistal myopathy (OPDMs). Although the upstream open reading frame has not been elucidated in OPML and OPDMs, polyglycine (polyG) translated by expanded CGG repeats is reported to be as a primary pathogenesis in FXTAS and NIID. Collectively, these findings indicate a new disease entity, the polyG diseases. In this review, we state the common clinical manifestations, pathological features, mechanisms, and potential therapies in these diseases, and provide preliminary opinions about future research in polyG diseases.

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.

Neuronal intranuclear inclusion disease (NIID) is a group of neurodegenerative diseases caused by GGC repeat expansion and is characterized by diverse clinical manifestations that may characterize a spectrum of underlying pathologies. Extensive inflammatory cell infiltration has been observed in multiple tissues obtained from NIID patients, including the temporal lobe, skin, lungs, colon, kidneys, and fallopian tubes. Intriguingly, eosinophilic intranuclear inclusions have been identified within the nuclei of these infiltrating inflammatory cells, underscoring the central role of inflammation in NIID. The infiltration of immune cells within the brain tissue of NIID patients highlights the critical role of neuroinflammation in disease pathogenesis. Moreover, in patients with NIID, not only is the neutrophil-to-lymphocyte ratio (NLR) markedly elevated but also the peripheral blood cytokine profile substantially altered. Despite the prominent involvement of central and peripheral inflammation in NIID and experimental evidence at the cellular level demonstrating that activation of the NF-κB‒NLRP3 pathway can reduce the burden of intranuclear inclusions and ameliorate phenotypic manifestations, therapeutic strategies targeting inflammation have yet to alter the disease course in human patients. This review presents the latest advancements in the study of inflammatory markers in NIID and the GGC100 cell model. Specifically, it explores the role of inflammatory molecular markers in disease pathogenesis and their correlation with clinical parameters. Furthermore, we summarize the potential applications of certain inflammatory biomarkers as novel therapeutic targets and strategies, which may pave the way for expanded therapeutic interventions in NIID.