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Abstract
Background NOTCH2NLC GGC repeat expansions have been associated with various neurogenerative disorders, including neuronal intranuclear inclusion disease and inherited peripheral neuropathies (IPNs). However, only a few NOTCH2NLC-related disease studies in IPN have been reported, and the clinical and genetic spectra remain unclear. Thus, this study aimed to describe the clinical and genetic manifestations of NOTCH2NLC-related IPNs.
Method Among 2692 Japanese patients clinically diagnosed with IPN/Charcot–Marie–Tooth disease (CMT), we analysed NOTCH2NLC repeat expansion in 1783 unrelated patients without a genetic diagnosis. Screening and repeat size determination of NOTCH2NLC repeat expansion were performed using repeat-primed PCR and fluorescence amplicon length analysis-PCR.
Results NOTCH2NLC repeat expansions were identified in 26 cases of IPN/CMT from 22 unrelated families. The mean median motor nerve conduction velocity was 41 m/s (range, 30.8–59.4), and 18 cases (69%) were classified as intermediate CMT. The mean age of onset was 32.7 (range, 7–61) years. In addition to motor sensory neuropathy symptoms, dysautonomia and involuntary movements were common (44% and 29%). Furthermore, the correlation between the age of onset or clinical symptoms and the repeat size remains unclear.
Conclusions These findings of this study help us understand the clinical heterogeneity of NOTCH2NLC-related disease, such as non-length-dependent motor dominant phenotype and prominent autonomic involvement. This study also emphasise the importance of genetic screening, regardless of the age of onset and type of CMT, particularly in patients of Asian origin, presenting with intermediate conduction velocities and dysautonomia.
- NEUROPATHY
- HMSN (CHARCOT-MARIE-TOOTH)
- NEUROGENETICS
- MRI
Data availability statement
Data are available upon reasonable request. Not applicable.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
NOTCH2NLC repeat expansions are associated with inherited peripheral neuropathy (IPN) as well as neuronal intranuclear inclusion disease.
WHAT THIS STUDY ADDS
This study demonstrates the comprehensive genetic spectrum of NOTCH2NLC repeat expansions based on the largest Japanese IPN case series to date, including approximately 2700 patients, and enhances our understanding of the clinical and pathological features of NOTCH2NLC-related diseases.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
Genetic screening of NOTCH2NLC repeat expansion should be performed in patients with IPN, regardless of the age of onset and type of Charcot–Marie–Tooth disease.
Introduction
Inherited peripheral neuropathies (IPNs) are a complex group of disorders that primarily affect the peripheral nervous system and demonstrate wide phenotypic and genotypic diversities. Charcot–Marie–Tooth disease (CMT) is the most common type of IPN, with a prevalence ranging from 9.7/100 000 to 82.3/100 000.1 Moreover, patients with CMT generally present with progressive motor sensory neuropathy, foot deformities and decreased tendon reflexes. CMT was traditionally classified according to median motor nerve conduction velocity (MNCV) and mode of inheritance, which is determined by family history. The major categories of CMT are demyelinating (median MNCV<38 m/s), axonal (median MNCV>38 m/s) and an intermediate form (median MNCV 25–45 m/s and preserved median compound muscle action potential) CMTs.2 3 Notably, IPN phenotypes, apart from CMT, include distal hereditary motor neuropathy (dHMN), hereditary sensory neuropathy, hereditary sensory autonomic neuropathy and hereditary neuropathy with liability to pressure palsy (HNPP). In the studies in the relevant literature, >140 genes have been associated with IPN/CMT to date.4 5 Moreover, comprehensive genetic analyses of IPN/CMT have been performed in several countries worldwide; however, a large proportion of patients remain genetically undiagnosed. For example, 66.7% of patients with IPN/CMT who have been screened for duplication/deletion of PMP22 remained undiagnosed in our previous report.5
In 2019, NOTCH2NLC GGC repeat expansion was identified as the cause of neuronal intranuclear inclusion disease (NIID)—a slowly progressive neurodegenerative disease with eosinophilic hyaline intranuclear inclusions in the central and peripheral nervous systems.6–8 The incidence of NIID is primarily high in Asian countries, including Japan and China; moreover, NIID has a wide range of neurologic phenotypes, including dementia, parkinsonism, tremor, cerebellar ataxia, seizures, peripheral neuropathy, myopathy and autonomic nervous system symptoms.9–13 Furthermore, recent studies have identified NOTCH2NC GGC repeat expansions in Taiwanese patients with IPN and in Chinese patients with dHMN and IPN.14–16 In this study, we screened NOTCH2NLC non-coding repeated expansion in >1700 genetically undiagnosed Japanese patients with IPN/CMT and describe the clinical and genetic characteristics of NOTCH2NLC-related diseases.
Material and method
Enrolment criteria
In this single-centre case series, we collected DNA samples of 2692 unrelated Japanese patients with IPN/CMT from 2007 to 2022 across various regions in Japan. All patients were examined by local neurologists, and their clinical data and blood/DNA samples were sent to our laboratory for genetic testing. All patients with the demyelinating CMT were confirmed to be negative for PMP22 duplication/deletion using fluorescence in situ hybridisation or multiplex ligation probe amplification.
Preliminary screening
First, gene panel sequencing was performed using DNA microarray or next-generation sequencing, targeting known/candidate IPN/CMT-related genes. Online supplemental table S1 shows the genes used in these examinations. Among patients who were negative at initial screening, 758 were further evaluated via whole-exome sequencing using the Illumina Hiseq platform (Illumina, San Diego, California, USA) or Ion Proton sequencing (ThermoFisher Scientific, Waltham, Massachusetts, USA). Subsequently, the remaining undiagnosed 1475 patients without autosomal dominant family history were screened for RFC1 repeat expansions using the previously described methodology.4 5 Figure 1A shows the study flowchart.
Supplemental material
Flowchart of NOTCH2NLC gene repeat analyses in the Japanese IPN/Charcot–Marie–Tooth disease case series. (A) Comprehensive preliminary genetic studies were performed in 2692 unrelated patients. The analysis included DNA microarray, next-generation sequencing-based gene panel sequencing, whole-exome sequencing and RFC1 analysis. NOTCH2NLC repeat expansions were identified from 22 of 1783 undiagnosed patients. (B) NOTCH2NLC repeat expansion is the seventh most frequent causative gene in the present Japanese case series. IPNs, inherited peripheral neuropathies; NGS, next-generation sequencing.
Repeat-primed PCR (RP-PCR) and fluorescence amplicon length analysis of NOTCH2NLC
The analysis included 1783 patients who were negative in preliminary screening. Furthermore, primer design, RP-PCR and fluorescence amplicon length analysis-PCR (AL-PCR) were performed according to a previous report.7 All RP-PCR products were subjected to capillary electrophoresis using the ABI PRISM 3130xL Genetic Analyzer (Applied Biosystems, Foster City, California, USA), and the results were visualised using the Peakscanner software (Applied Biosystems). For this study, GGC repeat size of ≧ 60 repeats was considered pathogenic.6–8 17 18 Online supplemental table S2 shows the primers used for RP-PCR and AL-PCR.
Statistical analysis
The Mann-Whitney U test and Pearson’s correlation tests were used to compare frequencies or numerical variables. A p values of <0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism V.9.3.1 (GraphPad Software, San Diego, California, USA).
Result
Analysis of repeat expansion in NOTCH2NLC
Pathogenic repeats in NOTCH2NLC were identified in 26 patients from 22 unrelated families among 1783 patients with IPN/CMT included in the present study. The median repeat size was found to be 146 (range, 71–222) (table 1). Online supplemental figure 1A shows examples of sawtooth patterns via RP-PCR and AL-PCR. Overall, NOTCH2NLC GGC repeat expansions were detected from 1.2% (22/1783) of genetically undiagnosed Japanese patients with IPN/CMT, and NOTCH2NLC GGC repeat expansion was the seventh most frequent causative gene in our case series, excluding the PMP22 copy number variations (figure 1B). Families with NOTCH2NLC pathogenic repeats were found spread over all eight regions of Japan, indicating a wide geographic distribution (online supplemental figure 2).
Clinical manifestation of 26 patients with NOTCH2NLC repeat expansions
Clinical summary
The clinical manifestations of all 26 patients from 22 families with NOTCH2NLC-related IPNs are presented in table 1. Eleven families had a history of NOTCH2NLC-related IPNs, and another 11 cases were sporadic. Online supplemental figure 1B shows the pedigrees of patients with a positive family history. However, segregation analysis was not available for the sporadic cases. In 26 patients with NOTCH2NLC-related IPNs, the mean age of onset was 32.7 years (range, 7–61 years). Moreover, all patients had absented or decreased deep tendon reflexes and most patients exhibited distal muscle weakness (24/26, 92%) and atrophy (20/23, 87%). Furthermore, sensory disturbance was present in approximately half of the patients (13/25, 52%). Apart from these symptoms of motor sensory neuropathy, dysautonomia was the most common symptom (11/25, 44%)—including neurogenic bladder, constipation, orthostatic hypotension, anhidrosis and erectile dysfunction. The involuntary movement was the next most common symptom (7/24, 29%)—including tremor (7/24, 29%), myoclonus (1/24, 4.2%) and fasciculation (1/24, 4.2%). Other less frequently identified symptoms were cognitive impairment (3/24, 13%), cerebellar ataxia (2/23, 9%) and pyramidal signs (1/23, 4%). Notably, none of the patients presented with parkinsonism (0/24, 0%). High serum creatine kinase (CK) levels were confirmed in 15 patients (15/20, 75%). The mean CK level was 559±445 IU/mL (range, 51–1681 IU/mL; normal range: 59–248 IU/mL in males and 41–153 IU/mL in females).
Table 2 demonstrated the result of nerve conduction studies and needle electromyography evaluated in the 26 patients. According to original and operational CMT classification based on median MNCV alone, 18 (69%) and 8 (31%) patients were grouped as axonal and demyelinating CMT, respectively. However, refining the phenotype further, 18 cases were classified as intermediate CMT and 8 axonal CMT. Electromyography revealed neurogenic changes in 12 cases (12/15, 80%) and no patients exhibited myogenic changes.
Electrophysiological findings in 26 patients with NOTCH2NLC repeat expansions
Radiological findings
Abnormal findings were observed in 13 of 16 patients who underwent MRI of the brain. Common findings included brain atrophy and/or leucoaraiosis, and most cases were of mild atrophy and/or leucoaraiosis. Only one patient (F14; MRI taken at 63 years of age) exhibited a U-fibre high signal on diffusion-weighted image, which is a characteristic finding of NIID. Figure 2 shows MRI findings of the brain of these patients.
Radiological findings of patients with NOTCH2NLC repeat expansions. Mild cerebral atrophy is observed in F1 (III-2), F1 (III-6) and F2, whereas leucoaraiosis or leucoencephalopathy is observed in F5, F8, F15, F17 (II-1) and F17 (II-2). Diffusion-weighted imaging abnormalities are observed only in F2 (spot-high lesion) and F14 (high U-fibre).
Pathological findings
Peroneus brevis muscle biopsy from F5 revealed chronic neurogenic changes (figure 3A,B). In this patient, sural nerve biopsy revealed decreased myelinated fibre density (3702/mm2) without demyelinated fibres or onion bulb formation (figure 3D–G), despite normal sensory nerve action potential. Immunostaining of the skeletal muscle and sural nerve specimens from F5 revealed p62-positive intranuclear inclusions (figure 3C–H), and electron microscopy of sural nerve revealed intranuclear inclusions with filamentous aggregates (figure 3I, J). Moreover, skin biopsy of the specimen from F14 revealed p62-positive intranuclear inclusions (figure 3K). Furthermore, sural nerve biopsy of the specimen from F15 revealed both demyelinating and axonal changes, without onion bulb formation (figure 3L).
Pathological findings of biopsy specimens from skeletal muscle, sural nerve and skin. (A–C) Peroneus brevis muscle from F5 shows neurogenic changes and p62 positive intranuclear inclusions. (A) H&E staining; (B) ATPase (pH=10.4), (C) Anti-p62 staining. (D–J) Sural nerve biopsy from F5 shows chronic axonal neuropathy and p62-positive intranuclear inclusions. (D,E) Toluidine blue staining; (F) Teased nerve fibre; (G) Histogram of myelinated fibre density, (H) Anti-p62 staining. Electron microscopy of the sural nerve ((I) perineurial cell, (J) Schwann cell) reveals intranuclear inclusions containing filamentous aggregates (white and black arrow). (K) Staining of skin biopsy specimen from F14 reveals p62-positive intranuclear inclusions. (L) Toluidine blue staining of the sural nerve biopsy specimen from F15 shows demyelinating and axonal changes, including thin myelin fibres, decrease in large and small myelinated fibre density and myelin ovoid.
Statistical analysis
No significant correlation was observed between the age of onset and repeat size (r=−0.08, p=0.71, Pearson’s correlation). Moreover, as shown in table 3 and online supplemental figure 3, the Mann-Whitney U test revealed no significant difference in repeat size between the patients with and without specific symptoms, including distal weakness (p=0.19), distal atrophy (p=0.22), sensory disturbance (p=0.31), cognitive impairment (p=0.37), dysautonomia (p=0.31), cerebellar ataxia (p=0.48), pyramidal signs (p=0.16), involuntary movement (p=0.32), miosis (p=1.00) and abnormal MRI findings of the brain (p=0.65).
The proportion of clinical manifestations and repeat number
Discussion
We identified pathogenic NOTCH2NLC repeat expansion in 22 unrelated families based on the analysis of the largest Japanese IPN/CMT case series to date, which included 1783 undiagnosed Japanese patients. This finding undoubtedly increased the rate of diagnosis and improved the genetic spectrum of our case series of patients with IPN/CMT. Moreover, these study findings clarified the clinical, electrophysiological, radiological and pathological features of patients with NOTCH2NLC-related diseases. NIID is a progressive neurodegenerative disease with various clinical manifestations attributed to NOTCH2NLC GGC repeat expansions.6–8 Moreover, NIID has a wide range of neurological phenotypes, including dementia, parkinsonism, tremor, cerebellar ataxia, seizures, peripheral neuropathy and autonomic nervous system symptoms. According to a study in 2005, the clinical presentations may also be confined to the peripheral nervous system, such as motor sensory and autonomic neuropathy.13 In 2021, NOTCH2NLC repeat expansion was identified in three Chinese families with distal motor neuropathy and rimmed vacuolar myopathy.19 In another study, NOTCH2NLC repeat expansions were identified in 7 patients with CMT2 in a Taiwanese IPN cohort of 127 patients, including 49, 66 and 12 cases of CMT, CMT2 and dHMN, respectively.14 In China, NOTCH2NLC repeat expansions were identified in 5 and 2 patients in a cohort of 142 unrelated patients with IPN and 90 unrelated families with dHMN, respectively.15 16 In the present study, NOTCH2NLC repeat expansion was present in 1.2% of patients with undiagnosed IPN/CMT, occupying the seventh most frequent causative gene in our Japanese case series (figure 1B). In summary, screening for NOTCH2NLC repeat expansion should be highly recommended for undiagnosed patients with IPN/CMT, particularly in Asian countries.
Our review of clinical records of all 26 patients with NOTCH2NLC repeat expansions revealed dysautonomia, involuntary movement, cognitive impairment, miosis, cerebellar ataxia and pyramidal sign in various combinations of symptoms in addition to neuropathic symptoms, such as distal weakness and atrophy, sensory disturbance and decreased deep tendon reflexes. Further, we summarised the clinical information of patients from previous IPN cohorts to understand the clinical diversity of NOTCH2NLC-related phenotypes (online supplemental table 3). The mean age of onset was similar in all studies, but the age of onset in some patients in our study was surprisingly less, that is, it was <10 years (1 case) or between 13 and 19 years (2 cases). Therefore, screening of NOTCH2NLC repeat expansion is necessary regardless of the age of onset. In the present study, muscle weakness and atrophy were more frequent than sensory disturbance, and dysesthesia was identified as an initial symptom in only one patient. Of the patients with NOTCH2NLC repeat expansion, muscle weakness was found in a distal-dominant form in 12 of the 22 patients (55%), whereas proximal and diffuse muscle weakness was observed in 2 (9%) and 8 (36%) of the 22 patients, respectively. No patient exhibited myogenic changes based on the results of electromyography or muscle biopsy, although the possibility of neuropathy and myopathy being simultaneously present could not be excluded. In summary, these findings suggest that NOTCH2NLC-related neuropathy may develop as a non-length-dependent motor dominant phenotype. Despite being a common symptom of NIID, parkinsonism was not identified in any patients in the current study. The symptoms of cerebellar ataxia and pyramidal signs were rare in the current case series; this finding is similar to those in previous reports.14 16 Dysautonomia—a frequent manifestation of NOTCH2NLC-related diseases—was found in 44% of patients in the current study. Conversely, dysautonomia was not found in the IPN in the Taiwanese study but was shared by all patients in the Chinese study. Notably, involuntary movement was a frequent symptom in the current case series as well as in the cohort of Chinese patients with IPN.16 Additionally, we identified NOTCH2NLC repeat expansion in one patient (F16) who exhibited HNPP-like episodes or recurrent compression neuropathy. The patient suddenly developed left peroneal nerve palsy at 55 years of age and achieved complete symptomatic recover without any disability after rehabilitation. However, the symptoms of the patient recurred at 56 years of age. Online supplemental table 4 shows the detailed electrophysiological findings of this patient. Moreover, in this patient, HNPP was suspected due to repeated nerve palsy, but PMP22 copy number variation was not found by fluorescence in situ hybridisation. Target resequencing of 72 genes, including PMP22, did not identify the causative gene. Therefore, to the best of our knowledge, this is the first study to report a case of NOTCH2NLC-related disease with HNPP-like phenotype or recurrent compression neuropathy. Additional case reports of patients with HNPP-like phenotypes are required to verify the association with NOTCH2NLC repeat expansion.
MRI abnormalities of the brain were more common in the present case series than in the two previous cohorts. However, in most cases in the present case series, the observed abnormalities were mild cerebral atrophy or mild leucoaraiosis, indicating that MRI findings of the brain are commonly normal or only mild abnormalities in patients with NOTCH2NLC-related diseases in the IPN cohort.
Electrophysiological analysis revealed that the median MNCV and median tibial MNCV were 41±7.3 (range, 30.8–59.4) and 34.7±5.7 (range, 21.0–54.9) m/s, respectively. The patients were classified as axonal (18 cases), and demyelinating (8 cases), where 18 cases could also be grouped as the intermediate form. In the Taiwanese and Chinese IPN cohorts, all cases were classified as axonal type with median nerve MNCV of >38 m/s, although most of these patients had slow tibial nerve velocity. Based on the criteria for intermediate CMT, six Taiwanese patients in previous studies could be classified as intermediate CMT.14 16 Thus, NOTCH2NLC repeat expansion analysis should be considered for all patients with IPN, especially for those with the intermediate CMT.
Previous studies investigated the relationship between GGC repeat size in NOTCH2NLC and variable phenotypes. Among patients with NIID, age of onset is less and repeats are longer in those with muscle weakness as the primary symptom (muscle weakness dominant type).7 8 However, GGC repeat size has no clear association with disease severity or onset.8 In the present study, there was no significant correlation between the repeat size and age of onset (p=0.71). Both fragile X syndrome (FXS) and fragile X-associated tremor/ataxia syndrome (FXTAS) are known to be caused by CGG triplet repeat expansions in the FMR1. Although fewer CGG repeats (50–200) cause FXTAS via a mechanism involving toxic RNA gain of function, longer CGG repeats (>200) cause FXS with loss of function.20 21 A similar mechanism might play a role in IPN and other phenotypes of NOTCH2NLC-related disease, although this is speculative. Otherwise, additional genetic, epigenetic or environmental factors might also contribute to the varied phenotypes of NOTCH2NLC-related disease.8 However, owing to technical and material limitations, we could not further examine these speculations in the current study. Other limitations of the present study include incomplete clinical examination by local hospitals and insufficient segregation analysis, particularly in patients without a family history. As a more accurate method for sizing repeats, southern blotting was not performed due to material and technical reasons, which could be a limitation for our genotype/phenotype analysis.
In conclusion, we identified NOTCH2NLC repeat expansions in 1.2% of patients without a previous genetic diagnosis of IPN/CMT in a large case series of 1783 patients. Moreover, clinical findings should facilitate the understanding of heterogeneity in NOTCH2NLC repeat expansions and the importance of genetic screening regardless of the age of onset and the type of CMT. Additionally, intermediate conduction velocity, autonomic dysfunction and involuntary movements were common in the current case series of patients with NOTCH2NLC-related IPN.
Data availability statement
Data are available upon reasonable request. Not applicable.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants. This study was approved by the institutional review board of Kagoshima University (Application ID: 490). Participants gave informed consent to participate in the study before taking part.
Acknowledgments
The authors thank all the patients and their families for participating in this study. The authors also thank Y Morita, A Tamura, T Nishihira, T Mizuno, K Sugie, N Iguchi, K Saigo, K Deguchi, T Tsuboi, N Miyaue, Y Matsumoto, K Sekiguchi, S Matoba, N Ono, T Ota, A Tsuneyama and K Kimura for clinical evaluation of the patients with NOTCH2NLC gene repeat expansion, and T Ohnishi at Kagoshima University for her great technical assistance. The authors are supported by Enago (www.enago.jp) for reviewing the English in this report. The authors extend their appreciation to the Division of Gene Research, Research Support Centre, Kagoshima University, for the use of their facilities.
References
Supplementary materials
Supplementary Data
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Footnotes
Twitter @pinetwitt
Contributors MA, YH and HT conceived the project and designed the study. MA, YH, JY and AY contributed to the analysis and interpretation of data. TH, FK, YN, MT, YH, SN, TN, YS and YO participated in analysis of clinical data. MD, AH, EM and JS contributed to pathological analysis. MA produced the original manuscript and all authors approved the final version. TH takes full responsibility for the overall content as the guarantor.
Funding This work was supported by Grants-in-Aid from the Research Committee of Ataxia, Health Labour Sciences Research Grant, the Ministry of Health, Labour and Welfare, Japan (201610002B). This work was also supported by the Research program for conquering intractable disease from Japan Agency for Medical Research and Development (201442014A, 201442071A and 17929553), JSPS KAKENHI Grant Numbers JP18H02742, JP20K16604, JP21K15702, JP21H02842, 15K09312, 19H03577 and MHLW FC program (JPMH19189624 and JPMH21445246).
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Competing interests None declared.
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