Essential tremor as the early symptom of NOTCH2NLC gene-related repeat expansion disorder

We read with great interest the recent article by Sun et al. titled ‘Expansion of GGC repeat in the human-specific NOTCH2NLC gene is associated with essential tremor’, which reported that abnormal GGC repeat expansion in the NOTCH2NLC gene was found in 11 families from a cohort study of 197 Chinese pedigrees clinically diagnosed with essential tremor (Sun et al., 2020). A recent work from the same group showed that expanded GGC repeats in the NOTCH2NLC gene cause neuronal intranuclear inclusion disease (NIID) and NIID-related diseases including Alzheimer disease and Parkinson disease (Tian et al., 2019). This raises a few questions: do expanded GGC repeats in the NOTCH2NLC gene cause different diseases? Or, do essential tremor-like, Alzheimer disease-like, and parkinsonism-like phenotypes belong to the subtypes of NIID? So far, NIID is considered a rare and slowly progressive neurodegenerative disease characterized by variable clinical manifestations including dementia, tremor, cerebellar ataxia, seizure, and peripheral neuropathy (Sone et al., 2016). Its pathological changes are eosinophilic intranuclear inclusions in the central and peripheral nervous systems, and visceral organs. Because of variable clinical manifestations, the diagnosis previously depended on typical neuroimaging with high-intensity signals in diffusion-weighted imaging (DWI) in the corticomedullary junction and NIID hallmarks such as eosinophilic hyaline intranuclear inclusions in brain tissues and/or skin (Sone et al., 2011, 2016; Chen et al., 2019). With genetic advances of NIID, GGC repeat expansion in the NOTCH2NLC gene was proposed to be the cause of NIID (Ishiura et al., 2019; Sone et al., 2019; Tian et al., 2019). Therefore, the presence of abnormal GGC repeats in the NOTCH2NLC gene could potentially serve as an important criterion for the diagnosis of NIID when differentiating from other genetic diseases. However, the study by Sun et al. (2020) challenged this and found that essential tremor is also associated with GGC repeats in the NOTCH2NLC gene. Moreover, Okubo et al. (2019) reported that GGC repeat expansion in the NOTCH2NLC gene in adult patients is associated with leukoencephalopathy. Therefore, to help address this question, we provide more evidence and clinical cases to examine the relationship between essential tremor and GGC repeat expansion in the NOTCH2NLC gene. In our cohort study, we recruited 15 families with clinically diagnosed familial essential tremor. For these families, we performed whole-exome sequencing to search for the causative genes and ruled out known genes for tremor. As tremor is a common symptom of NIID, we examined GGC repeats in the NOTCH2NLC gene in patients with familial essential tremor via a repeat-primed PCR method as described previously (Sone et al., 2019). Interestingly, abnormal GGC repeat expansion in the NOTCH2NLC gene was found in one family with familial essential tremor as in Fig. 1A. The proband (Patient III4) was a 67-year-old male patient who complained of tremor in head and both hands for 4 years. Physical examination revealed no positive signs except a kinetic tremor as depicted in Supplementary Video 1. Brain MRI showed no obvious white matter abnormality (Fig. 1B). Because several family members had a similar pattern of tremor, the diagnosis of familial essential tremor was made. However, 4 years later, Brain MRI showed abnormal DWI as shown in Fig. 1C. Esophageal polyp biopsy showed p62-positive cells and ubiquitin-positive cells by immunohistochemistry staining, indicating the existence of intranuclear inclusion (Fig. 1D and E). Therefore, NIID was suspected, and abnormal GGC repeat expansion in the NOTCH2NLC gene was further confirmed in this patient (Fig. 1F). In this family, there were 16 individuals with abnormal GGC repeat expansion and 10 individuals among them had a similar tremor. In these 10 patients, the median age of onset was 53.5 (interquartile range: 48, 60) years, and median duration was 7.5 (interquartile range: 4.5, 11.5) years. The tremor sites were the upper limbs and head. The score of the Essential Tremor Rating Assessment Scale was 10.3 ± 8.0. Recent follow-up revealed that four patients had worsened cognitive functions and the average (standard deviation) score of the Mini-Mental State Examination in 10 patients was 19.5 ± 6.8. The proband’s cousin (Patient III10) had typical bilateral ribbon-like high-intensity signals along the corticomedullary junction on brain MRI (Fig. 1G). The brain MRI of the proband’s younger sister (Patient III6) with typical kinetic tremor showed no leukoencephalopathy (Fig. 1H); however, her skin biopsy showed p62-positive cells (Fig. 1I) and ubiquitin-positive cells (Fig. 1J). Considering family history, symptoms such as tremor and dementia, typical MRI, genetic evidences, and pathological staining, this family was diagnosed with NIID. An essential tremor family with abnormal GGC repeat expansion in the NOTCH2NLC gene. (A) A pedigree of familial essential tremor is shown. Filled black symbols indicate individuals with GGC repeat expansion; filled grey symbols indicate suspected patients without genetic tests. (B) The proband had normal brain MRI at the age of 63 years. (C) Brain MRI revealed abnormal high-intensity signals in the corticomedullary junction in DWI at the age of 67 years of the proband. (D and E) Immunohistostaining showed anti-p62 cells (D) and anti-ubiquitin (UB, E) cells in the oesophageal polyp of the proband. (F) Electropherogram of repeat-primed PCR showed no GGC repeat expansion in the NOTCH2NLC gene in unaffected individual (top) and a saw-tooth tail pattern of the GGC repeat expansion in the NOTCH2NLC gene of the proband (bottom). (G and H) Brain MRI showed typical bilateral ribbon-like high-intensity signals along the corticomedullary junction in a patient (Patient III10) with slight tremor (G); however, no abnormal MRI changes were found in a patient (Patient III6) with typical essential tremor (H). (I and J) Immunohistostaining showed anti-P62 cells (I) and anti-ubiquitin (J) cells in the skin of Patient III6. The increasing line of evidence demonstrates that NIID is a heterogeneous disease with regard to age at onset, clinical symptoms, and brain imaging features. Age at onset ranges widely from infantile to advanced age (Takahashi-Fujigasaki, 2003; Sone et al., 2016). Heterogeneity of clinical symptoms has been extensively reported, including cognitive dysfunction, parkinsonism-like behaviours, peripheral neuropathy, ataxia, tremor, involuntary movement, muscle weakness, seizure, and headache (Sone et al., 2016; Takahashi-Fujigasaki et al., 2016; Tian et al., 2019; Wang et al., 2020). For brain MRI, although some cases showed characteristic zigzag edging sign along the corticomedullary junction on DWI, more patients had atypical leukoencephalopathy and lesions in different brain regions including corticomedullary junction, corpus callosum, and cerebellum (Abe and Fujita, 2017; Sugiyama et al., 2017; Kawarabayashi et al., 2018). Therefore, variable clinical manifestations and MRI changes render the diagnosis of NIID difficult. In our pedigree, so far, 10 patients have presented with essential tremor-like symptoms. Some patients showed no abnormal brain imaging at disease onset; however, with disease development, patients revealed typical zigzag edging sign along the corticomedullary junction on brain MRI as previously described (Chen et al., 2019). In addition, we also provided pathological evidence to show anti-p62 and anti-ubiquitin-positive cells (two hallmarks of intranuclear inclusion) in skin and oesophageal polyp tissues. Combined with brain MRI, pathological changes, and genetic evidence of GGC repeat expansion in the NOTCH2NLC gene, NIID was diagnosed in this family, indicating that essential tremor is an early symptom of NIID or NOTCH2NLC gene-related repeat expansion disorders. Although Sun et al. showed that essential tremor is highly associated with GGC repeat expansion in the NOTCH2NLC gene, long-term follow-up is needed to rule out the diagnosis of NIID. It is unclear whether the length of GGC repeat expansion in the NOTCH2NLC gene results in distinct pathogenic mechanisms and causes the heterogeneity of clinical symptoms; this enigma warrants further investigation. Additionally, the highly heterogeneous clinical symptoms of NIID indicate that disease modifying factors may exist and should be further defined in NOTCH2NLC gene-related repeat expansion disorders. The data that support the findings of this study are available from the corresponding author, upon reasonable request. This work was supported by the grants from National Key R&D Program of China (2017YFE0103700 and 2019YFA0802600), the National Science Foundation of China (81120108011, 81771454, and 81974244), and the Priority Academic Program Development of Jiangsu Higher Education Institutions of China. The authors report no competing interests.