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NOTCH2NLC-related disorders: the widening spectrum and genotype–phenotype correlation
  1. Yu Fan1,2,
  2. Yuming Xu1,3,4,
  3. Changhe Shi1,3,4
  1. 1 Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, People's Republic of China
  2. 2 Academy of Medical Sciences of Zhengzhou University, Zhengzhou, Henan, People's Republic of China
  3. 3 Henan Key Laboratory of Cerebrovascular Diseases, Zhengzhou University, Zhengzhou, Henan, People's Republic of China
  4. 4 Institute of Neuroscience, Zhengzhou University, Zhengzhou, Henan, People's Republic of China
  1. Correspondence to Dr Changhe Shi, Department of Neurology, Zhengzhou University First Affiliated Hospital, Zhengzhou, China; shichanghe{at}gmail.com

Abstract

GGC repeat expansion in the 5′ untranslated region of NOTCH2NLC is the most common causative factor in neuronal intranuclear inclusion disease (NIID) in Asians. Such expanded GGC repeats have been identified in patients with leukoencephalopathy, essential tremor (ET), multiple system atrophy, Parkinson’s disease (PD), amyotrophic lateral sclerosis and oculopharyngodistal myopathy (OPDM). Herein, we review the recently reported NOTCH2NLC-related disorders and potential disease-causing mechanisms. We found that visual abnormalities may be NOTCH2NLC-specific and should be investigated in other patients with NOTCH2NLC mutations. NOTCH2NLC GGC repeat expansion was rarely identified in patients of European ancestry, whereas the actual prevalence of the expansion in European patients may be potentially higher than reported, and the CGG repeats in LRP12/GIPC1 are suggested to be screened in European patients with NIID. The repeat size and interruptions in NOTCH2NLC GGC expansion confer pleiotropic effects on clinical phenotype, a pure and stable ET phenotype may be an early symptom of NIID, and GGC repeats in NOTCH2NLC possibly give rise to ET. An association may also exist between intermediate-length NOTCH2NLC GGC repeat expansion and patients affected by PD and ET. NOTCH2NLC-OPDM highly resembles NOTCH2NLC-NIID, the two disorders may be the variations of a single neurodegenerative disease, and there may be a disease-causing upper limit in size of GGC repeats in NOTCH2NLC, repeats over which may be non-pathogenic. The haploinsufficiency of NOTCH2NLC may not be primarily involved in NOTCH2NLC-related disorders and a toxic gain-of-function mechanism possibly drives the pathogenesis of neurodegeneration in patients with NOTCH2NLC-associated disorders.

  • genetics
  • genotype
  • phenotype
  • DNA repeat expansion

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Introduction

The clinical phenotypes associated with the GGC repeat expansion in the 5′ untranslated region (UTR) of NOTCH2NLC are highly variable. It was initially identified in patients with neuronal intranuclear inclusion disease (NIID), a slowly progressing neurodegenerative disease characterised by the formation of eosinophilic intranuclear inclusions in the nervous system and multiple visceral organs. Studies noted the presence of other clinical phenotypes in probands with expanded NOTCH2NLC GGC repeats and their family members with a higher frequency than would be expected by chance. The variability in the clinical presentation of NOTCH2NLC-related disorders is a challenge and a hope, as it suggests that more than one disease modifiers are at work, each of which is a potential therapeutic target. Repeat size and interruptions in expanded GGC repeats are the prominent modifiers; however, their role has not been explicitly understood in NOTCH2NLC-related disorders.

NOTCH2NLC-related disorders

NOTCH2NLC-NIID

NIID is a multisystem neurodegenerative disease characterised by the deposition of eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. The clinical manifestations of NIID are highly variable, including muscle weakness, parkinsonism, cerebellar ataxia, dementia, neuropathy, autonomic dysfunction, and seizure. Since dementia and limb weakness are the two main symptoms of NIID, it is categorised as dementia-dominant and limb-weakness-dominant subtypes. The multisystem symptoms of NIID could be explained by the wide distribution of intranuclear inclusions in the central nervous system and other organs. The deposition of ubiquitin-positive and p62-positive intranuclear inclusions in the skin, a symmetrical high-intensity signal in the corticomedullary junction on diffused weight image (DWI), and a remarkable leukoencephalopathy observed in the MRI support a NIID diagnosis.1

The first patient with NIID was reported in 1968.2 Because of the wide range of clinical phenotypes and challenges in diagnosis, the number of reported NIID cases was limited to about 40 cases till 2011. The number of patients diagnosed with NIID substantially increased worldwide with the advent of the method by Sone and colleagues3 that diagnoses NIID using skin biopsy. They have established a diagnostic flowchart for adult-onset NIID.1

Recently, GGC repeat expansion in the 5′ UTR of NOTCH2NLC was identified as the most common causative factor of NIID.4–9 The GGC repeat expansion accounts for nearly 100% of NIID cases in China4 7 and Japan5 6 but almost nil in the European population.9 So far, the only two European patients with NIID (diagnosed with the European NIID criteria proposed by Chen and co-workers9) harbouring NOTCH2NLC GGC repeat expansion were identified both in the Ukrainian population using in silico screening of whole-genome sequencing data in a large population cohort (50 544 patients in total),9 10 suggesting distinct genetic architecture of Asian and European patients with NIID. In addition, NOTCH2NLC GGC repeat expansion was found extremely rare in European patients with leukoencephalopathy,11 essential tremor (ET),12 Parkinson’s disease (PD),13 spinocerebellar ataxia10 and multiple system atrophy (MSA),10 suggesting the geographical heterogenicity of NOTCH2NLC GGC repeat expansion. The variable frequency of NOTCH2NLC GGC repeat expansion worldwide is similar to the GGGGCC repeat expansion in C9ORF72. Therefore, there might be a common founder effect responsible for the geographical heterogenicity of repeat expansion in NOTCH2NLC, resembling the GGGGCC repeats in C9ORF72. The origin of NOTCH2NLC GGC repeat expansion warrants further investigation to help explain the geographically heterogeneous carrier frequency.

Details of NOTCH2NLC-NIID and non-NOTCH2NLC-NIID patients reported so far are summarised in online supplemental table 1. Within the broad range of clinical phenotype, visual abnormalities (miosis, night blindness, reduced electroretinographic responses and degenerative retinal changes) are more frequent in patients with NOTCH2NLC-NIID as 34.1% (29/85) of patients with NOTCH2NLC-NIID reported visual abnormalities, but none in non-NOTCH2NLC-NIID patients, suggesting that the visual abnormalities may be NOTCH2NLC-specific. Patients with NOTCH2NLC-NIID presented with similar ophthalmologic features as rod-cone dysfunction and progressive retinal degeneration in peripapillary and mid-peripheral regions.14 Given the low incidence of NOTCH2NLC-NIID, retinal dystrophy or other ophthalmologic abnormalities, these rare disorders are unlikely to appear simultaneously in an individual.15 Interestingly, despite the rareness of NOTCH2NLC GGC repeat expansion in European patients with NIID, one of the only two reported European patients with NOTCH2NLC-NIID showed hemi-visual disturbance, visual aura, vertigo and hemi-visual loss,10 further emphasising the specificity of visual abnormalities in NOTCH2NLC-NIID. Further studies on visual function in such patients are warranted to elucidate this concern. Moreover, detailed ophthalmological examinations and retinal biopsies are recommended in subjects harbouring NOTCH2NLC GGC repeat expansion for an early diagnosis of the probable NOTCH2NLC-retinopathy.

Supplemental material

A higher positive rate of skin biopsies was identified in NOTCH2NLC-NIID. However, only 14 non-NOTCH2NLC-NIID patients have been reported so far, with a detailed description of 11 of them not available. Chen and co-workers9 reported an earlier age at onset and a much lower antemortem skin biopsy diagnosis rate in non-Japanese patients with NIID compared with Japanese patients with NIID (9% and 74%, respectively), suggesting less extraneuronal involvement in the former. Intriguingly, a comparison of inclusions in Japanese and European patients with NIID showed that the inclusions are fine granulose in the former but filamentous in the latter.

Additionally, although rare, a portion of Japanese patients with NIID with supporting pathological findings were found not harbouring expanded GGC repeats in NOTCH2NLC.5 This evidence suggests a genetically heterogeneous mechanism underlying the non-NOTCH2NLC-NIID. Screening for repeat expansion of GGC or other motifs in other genes is needed to explore the genetic basis of non-NOTCH2NLC-NIID.

Studies have identified the length of GGC repeats as a main impact factor of phenotype in patients with NOTCH2NLC-NIID, as the largest GGC repeat expansion (118–517 repeats) might lead to muscle weakness-dominant phenotype, a shorter (91–268 repeats) expansion might result in dementia-dominant phenotype and a much shorter repeat expansion (66–102 repeats) might cause parkinsonism-dominant phenotype.4 Interestingly, a patient with familial dementia-dominant NIID was identified harbouring a biallelic GGC repeat expansion (60 and 76 repeats), with no detailed data given, leaving the association of a biallelic shortly expanded GGC repeats in NOTCH2NLC with a more severe clinical phenotype elusive.6 In addition, the interruptions in GGC repeats were found to modify the age of onset,16 clinical manifestations17 and somatic stability.18 An expanded GGC repeats with GGA interruptions instead of a pure GGC repeat expansion may cause a muscle weakness-dominant phenotype and an earlier age at onset.6 8 However, whether and how interruptions modify the NIID clinical phenotype remains elusive.

NOTCH2NLC-ET

Tremor is one of the main clinical manifestations of NIID.4 5 7 ET is a common movement disorder affecting both children and adults with high heritability and prevalence that increases dramatically with ageing. ET is defined clinically as isolated 4–12 Hz kinetic tremors of the arms for at least 3 years that also affects the neck, vocal cords and other locations.19 Patients with ET with neurological symptoms, such as cognitive decline, psychiatric disorders, and hearing loss, are categorised as ‘ET plus’. Utilising a comprehensive strategy, Sun and co-workers reported ET as a second disorder that might be associated with GGC repeat expansion in the 5′ UTR of NOTCH2NLC.20

NOTCH2NLC GGC repeat expansion accounts for 5.6% (11/197 pedigrees) familial ET in the cohort of Sun et al, the 27 patients with familial NOTCH2NLC-ET from the 11 genetically positive pedigrees harboured 60–250 GGC repeats, whereas their unaffected familial members and over 1300 healthy controls harboured 4–41 GGC repeats. In NOTCH2NLC-ET, the tremor phenotype was more severe, the activities of daily living ability were worse and voice was more frequently involved.14 The repeat size was shorter and the frequency of GGA interruptions was lower in NOTCH2NLC-ET than those in NIID.20 21 Additionally, skin biopsies showed deposition of ubiquitin-positve and p62-positve intranuclear inclusions in three genetically positive ET probands but not in a non-NOTCH2NLC-ET proband.14 However, Sone and co-workers3 have verified the deposition of intranuclear inclusions in dermal cells as a useful tool for diagnosis and differential diagnosis of NIID.

This study is of great significance, as it might reveal a new candidate genetic risk factor that underly ~6% familial patients with ET.14 NOTCH2NLC GGC repeat expansion was later screened in Chinese,22 23 Singaporean21 and European10 12 24 patients affected by ET and found to account for 0%–6.67% ET pedigrees/patients. Nevertheless, some patients with NOTCH2NLC-ET were suspected or eventually diagnosed with NIID based on their family history, symptoms, MRI, genetic evidence, and most importantly, the positive pathological staining.10 21–23

Following are the most significant concerns: are such patients with NOTCH2NLC-ET actually affected by NIID? Are NOTCH2NLC-ET and NIID two distinct disorders that coincidentally attribute to NOTCH2NLC GGC repeat expansion? Genetic pleiotropy of GC-rich repeat expansion has been verified in the CGG repeats in the FMR1 gene. Over 200 CGG repeats called ‘full mutation’ lead to FMR1 silencing and fragile X syndrome (FXS), whereas the 55–200 CGG repeats called ‘premutation’ result in an incredible FMR1 mRNA upregulation, RNA gain-of-function toxicity and fragile X-associated tremor/ataxia syndrome (FXTAS).25 FXS and FXTAS are two separate disorders with distinct molecular aetiologies affecting different patient groups, despite both having CGG repeat expansion in FMR1.

It is plausible that NOTCH2NLC GGC repeat expansion indeed gives rise to ET. The 27 clinically diagnosed patients with NOTCH2NLC-ET in the cohort of Sun et al presented with simple kinetic and/or postural tremor without dementia, ataxia, peripheral neuropathy and hypermyotonia. MRI revealed no severe leukodystrophy and cerebellar atrophy. All of them had a long disease course, and nearly half of them had the disease for more than 10 years. The only presentation that challenged their ET diagnosis was the positive findings of skin biopsies.20 The 27 patients with NOTCH2NLC-ET were unable to be diagnosed with NIID according to the diagnosis flowchart of adult-onset NIID proposed by Sone and colleagues,1 highlighting that NOTCH2NLC-ET might be classified within NIID-related disorders (NIIDRDs), a disease group with formation of neuronal intranuclear inclusions, instead of NIID. Notably, such patients may present with a long-term and stable pure ET phenotype even for 10 years before progressing to NIID.21 22 Taken together, NOTCH2NLC GGC repeat expansion may be associated with ET, and a pure ET phenotype may be the early stage of NIID.22 A different repeat size and frequency of interruptions may modify the clinical presentation and lead to a distinct phenotype.

Yau and co-workers10 identified one patient with recurrent encephalopathy and postural tremor/parkinsonism harbouring NOTCH2NLC GGC repeat expansion in the 100 000 Genomes Project, revealing an extremely low prevalence of NOTCH2NLC GGC repeat expansion in European patients with movement disorders. In their study, some cases with interrupted GGC repeats and low reads number were regarded as ‘false-positives’ and excluded. However, the interruptions are common in expanded GGC repeats in NOTCH2NLC and other genes,6 20 21 26 which are thought to modify the age at onset and clinical presentation,16 21 and reads with repeat expansion were detected less frequently than those without expansion.21 27 28 Moreover, using ExpansionHunter, Yau et al identified 10 patients with neurological symptoms harbouring over 40 GGC repeat expansion in another study.9 This evidence implies that the actual prevalence of NOTCH2NLC repeat expansion in European patients with movement disorders and neurological symptoms may be higher than reported.

NOTCH2NLC-PD

PD is a common neurodegenerative disease characterised by bradykinesia, rest tremor, rigidity, gait disturbance and progressive loss of neurons in substantia nigra.29 Some patients with NIID presented with parkinsonism, tremor, and other extrapyramidal symptoms.4 30

Tian and colleagues4 screened NOTCH2NLC GGC repeat expansion in a familial parkinsonism cohort and identified 3/205 (1.5%) of parkinsonism-affected families harbouring the expanded GGC repeats in NOTCH2NLC. Their skin biopsies revealed the deposition of ubiquitin-positive and p62-positive intranuclear inclusions. The high-intensity signal along the corticomedullary junctions on DWI and the severe leukoencephalopathy were shown in 1/6 and 2/6 parkinsonism-affected cases, respectively. The three families were then diagnosed with parkinsonism-dominant NIID. Tian and co-workers divided familial genetically positive NIID cases into three subgroups based on their initial and main clinical features, and they observed a much smaller repeat size in parkinsonism-dominant NIID (66–102 repeats) than in muscle weakness-dominant NIID (118–517 repeats) and dementia-dominant NIID (91–268 repeats).

Intriguingly, as would be expected from the small repeat size identified in parkinsonism-dominant NIID, the GGC expansion ranged 41–64, which was termed as the ‘intermediate-length’ repeat expansion, was independently identified in 1.00%–1.09% sporadic PD cases in Asians (both p<0.001). All the patients with NOTCH2NLC-PD exhibited typical PD manifestations and responded to small dosages of levodopa. Some patients who were followed up for many years did not progress to clinical or radiological features of NIID.28 31 Shi and co-workers31 identified no patients with PD harbouring over 52 GGC repeats in their cohort. Skin biopsies of two patients with PD harbouring intermediate-length GGC repeat expansion showed deposition of phosphorylated alpha-synuclein instead of ubiquitin-positive and p62-positive intranuclear inclusions, pathologically emphasising their diagnosis of PD instead of the NIID. Moreover, a higher GGC frequency and a lower frequency of GGA/AGC interruptions were observed in patients with intermediate-NOTCH2NLC-PD than in patients with NIID,31 reminiscent of the lower frequency of GGA interruptions identified in the patients with NOTCH2NLC-ET than in patients with NIID.20 21 In addition, Ma and colleagues28 identified three patients with PD harbouring over 64 GGC repeats. No GGA interruptions was found; however, a three-time higher frequency of AGC interruptions was identified in these three patients. The data of skin biopsies of the three patients were not shown, leaving the pathological diagnosis elusive.

Taken together, evidence suggests that the intermediate-length GGC repeat expansion in NOTCH2NLC and a low frequency of GGA interruptions may be associated with PD instead of the parkinsonism-dominant NIID. Yau and co-workers identified no expansion carrier in European PD cohort,10 13 highlighting further the rareness of NOTCH2NLC GGC repeat expansion in the European population.

There were no studies investigating the NOTCH2NLC intermediate-length GGC repeat expansion in any human neurological disorders until Ma et al 28 and Shi et al 31 reported that patients with PD could harbour intermediate-length GGC expansion in NOTCH2NLC. Moreover, Ng et al 21 and Yan et al 23 have previously reported patients with ET harbouring 47–53 and 41–48 GGC repeats; however, the detailed data were not provided. Put together, it is plausible that the intermediate-length NOTCH2NLC GGC repeat expansion confers a higher risk of PD and ET in the Asian population. In addition, patients affected by Alzheimer’s disease (AD),32 leukoencephalopathy, corticobasal syndrome (CBS) and progressive supranuclear palsy (PSP)33 harbouring 41–64 GGC repeats have also been reported. Further studies are needed to explore the role of NOTCH2NLC intermediate-length GGC repeat expansion in these disorders.

Unlike the large NOTCH2NLC GGC expansion in NIID and ET, the two patients with intermediate-NOTCH2NLC-PD showed no deposition of ubiquitin-positive and p62-positive intranuclear inclusions,31 suggesting that the intermediate-length NOTCH2NLC GGC repeats may not be associated with the aggregation of repeat-associated non-AUG (RAN) translated protein, resembling the intermediate-length repeat expansion in other genes.34 Therefore, the patients affected by intermediate-NOTCH2NLC-PD and NOTCH2NLC-ET may be classified within NOTCH2NLC-related disorders but not NIIDRD or NIID. The mechanisms by which the intermediate-length and large GGC repeat expansion leads to distinct clinical phenotype warrant further investigation.

Taken together, NOTCH2NLC joins a repeat expansion-containing gene list in which the repeat size confers a pleiotropic effect on clinical phenotype (figure 1). For instance, the large GGGGCC expansion in C9ORF72 causes amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD),35 whereas the intermediate-length expansion confers a 3.5-fold higher risk of corticobasal degeneration.34 Similarly, the intermediate-length CGG repeat expansion in FMR1 results in FXTAS linked to neurotoxicity of repeat RNA, whereas the full expansion leads to FXS because of complete FMR1 silencing.25 For Ataxin-2, the full CAG expansion causes SCA2,36 whereas the intermediate-length expansion is a risk factor for ALS.37

Figure 1

Length of the GGC repeat expansion identified in NOTCH2NLC-related disorders. The patients affected by parkinsonism-dominant NIID and parkinsonism-like phenotype (eg, PD, ET, PSP, CBS) harboured the shortest GGC repeat expansion and the narrowest range; the patients affected by dementia-dominant NIID and AD harboured a longer GGC expansion and a wider repeat range, whereas the patients affected by muscle weakness-dominant NIID and muscle weakness-related disorders (eg, OPDM) harboured the largest GGC repeat expansion and an extremely wide range. Evidence shows a tendency that an expanded GGC repeats with more GGA interruptions may cause a muscle weakness-dominant NIID phenotype; with fewer GGA interruptions may cause ET phenotype; with non-GGC/GGA/AGC/ACG interruptions may lead to OPDM phenotype; and a much shorter expanded GGC repeats interrupted by fewer GGA repeats may cause PD phenotype. The correlation between phenotype with interruptions is not clear, further work is needed to explore the role of interruptions in the heterogeneity of NOTCH2NLC-related disorders. AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; CBS, corticobasal syndrome; ET, essential tremor; MSA, multiple system atrophy; NIID, neuronal intranuclear inclusion disease; OPDM, oculopharyngodistal myopathy; PD, Parkinson’s disease; PSP, progressive supranuclear palsy.

NOTCH2NLC-OPDM

Oculopharyngodistal myopathy (OPDM) is a clinically and genetically distinct autosomal-dominant myopathy characterised by ptosis, external ophthalmoplegia, weakness of masseter, facial, pharyngeal and distal limb muscles.38 Given the similarity in clinical and radiological findings of NIID and OPDM, non-coding GGC/CGG repeat expansion was screened in OPDM cohorts, identifying the GGC/CGG repeat expansion in the 5′ UTR of LRP12 and GIPC1 accounts for 54.17% and 28.61% of Asian OPDM cases,5 39 respectively.

Recently, two studies identified the large GGC repeat expansion in the 5′ UTR of NOTCH2NLC as the third causative factor of OPDM,40 41 which accounts for 16.67% of patients with OPDM in the Chinese population. In addition to the typical OPDM clinical manifestations, patients affected by NOTCH2NLC-OPDM showed leukoencephalopathy, retinal pigmentary degeneration, tremor, ataxia, peripheral neuropathy, increased cerebrospinal fluid (CSF) protein and serum creatine kinase levels, and other abnormalities in the central or peripheral nervous system.40 41 The aberrant findings of DWI, MRI, nerve conduction studies, CSF protein elevation and skin biopsies in some patients with NOTCH2NLC-OPDM highly confused the definitive diagnosis.40 41 In addition, the frequency of non-GGC/GGA/AGC/ACG repeat interruptions in NOTCH2NLC-OPDM was found higher than that in NIID.41 Interestingly, when reviewing NIID, we suggested that the visual abnormalities may be NOTCH2NLC-specific, and given that all patients with NOTCH2NLC-OPDM showed retinal pigmentary degeneration, a non-typical OPDM clinical manifestation, the evidence further corroborates our finding.

Diagnostically, the deposition of ubiquitin-positive and p62-positive intranuclear inclusions in the skin, nervous systems and other organs are pathognomonic of NIID.1 Ogasawara and co-workers40 identified the deposition of p62-positive intranuclear inclusions in 2/7 patients with NOTCH2NLC-OPDM, who would be diagnosed with NIID according to the proposed NIID diagnostic flowchart.1 However, the ubiquitin-positive and p62-positive intramyonuclear inclusions, which essentially are the same as intranuclear inclusions in the neurons, were widely identified in the myonuclear centre of all seven patients with NOTCH2NLC-OPDM.40 The study by Yu et al 41 further corroborated the observation by Ogasawara et al. Moreover, patients affected by NOTCH2NLC-OPDM showed additional peripheral and/or central nervous system involvement, which fills the phenotypic gap between NIID and OPDM. Thus, two key questions should be posed: are the ubiquitin-positive and p62-positive inclusions depositing in different organs (eg, muscle, skin, central nervous system) and a variable affected degree that drives the distinct but highly overlapping clinical, pathological and radiological manifestations of NIID and OPDM? Are OPDM and NIID both variations of a single neurodegenerative disease?40

NOTCH2NLC was identified as the third causative gene of OPDM,41 following LRP12 5 and GIPC1.39 Chen and colleagues9 identified no NOTCH2NLC expansion carrier in European patients with NIID, claiming that NIID is genetically heterogeneous. Given that patients with NOTCH2NLC-OPDM showed deposition of ubiquitin-positive and p62-positive intranuclear inclusions both in the skin and in the muscles40; patients with LRP12-OPDM showed ubiquitin-positive and p62-positive intranuclear inclusions in central and peripheral nervous systems,42 and patients with GIPC1-OPDM showed deposition of p62-positive inclusions in the muscle nuclei, whether the GGC/CGG repeat expansion in LRP12/GIPC1 contributes to the pathogenesis of NIID in European population needs further investigation.

Interestingly, a patient with OPDM harbouring a biallelic NOTCH2NLC GGC repeat expansion (674 and 217 repeats) had even a milder clinical phenotype than a patient harbouring a monoallelic GGC repeat expansion (139 repeats) who showed an early-onset and severe OPDM symptoms, suggesting a complex correlation underlying NOTCH2NLC GGC repeats and related disorders. Moreover, Yu and co-workers41 reported two asymptomatic individuals (families of affected individuals) both harbouring over 400 NOTCH2NLC GGC repeats showed neither NIID nor OPDM manifestations. No abnormalities in the brain and muscle MRI, nerve conduction studies, electromyography, and skin or muscle biopsies were found, suggesting that individuals harbouring a much higher GGC expansion may be totally but paradoxically asymptomatic.41

NOTCH2NLC-MSA

MSA is an adult-onset, fatal neurodegenerative disorder, clinically characterised with various combinations of symptoms such as parkinsonism, progressive autonomic failure, cerebellar and pyramidal features,43 and pathologically featured with abnormal accumulation of misfolded alpha-synuclein in oligodendrocytes.44 NIID has highly variable clinical manifestations, including parkinsonism, autonomic dysfunctions, ataxia, pyramidal signs, dementia and peripheral neuropathy.1 In addition, neuronal intranuclear hyaline inclusions have been identified in cases affected by MSA.45 46

Recently, Fang and colleagues27 identified 2.6% (5/189) patients with MSA harbouring the NOTCH2NLC GGC repeat expansion (no statistically significant difference was identified), wherein skin biopsies of 2/5 patients with NOTCH2NLC-MSA revealed p62-positive but alpha-synuclein-negative eosinophilic inclusions. One patient showed mild high-intensity signal along the corticomedullary junction on DWI, and the other four patients showed white matter hyperintense, which was thought unrelated to the NOTCH2NLC GGC repeat expansion.27 The MSA phenotype in patients harbouring NOTCH2NLC GGC repeat expansion is representative of the whole clinical spectrum of MSA. The median survival time of MSA is 6–9 years from symptoms onset,47 whereas patients with NIID have a much longer disease duration than patients with MSA. Interestingly, the disease duration has been found to be longer in NOTCH2NLC-MSA patients than in non-NOTCH2NLC-MSA patients, suggesting that NOTCH2NLC-MSA progresses much more slowly than non-NOTCH2NLC-MSA. In addition, evidence shows a higher incidence of severe urinary retention in NOTCH2NLC-MSA compared with non-NOTCH2NLC-MSA; all five patients with NOTCH2NLC-MSA showed bladder dysfunction-dominated autonomic dysfunctions, and urinary retention was the most frequent issue in these patients. As would be expected from the NIID clinical manifestations, there is also a greater incidence of prominent cognitive impairment in NOTCH2NLC-MSA.27 However, in another Chinese cohort with 328 patients with MSA, no NOTCH2NLC GGC repeat expansion was identified.48 Screening of NOTCH2NLC GGC repeat expansion in a European MSA cohort also failed to identify any expansion carriers, in which 336 patients with pathologically confirmed MSA were included.10 The role of NOTCH2NLC GGC repeat expansion in MSA needs further investigation.

NOTCH2NLC-ALS

ALS is a relentlessly progressive neurodegenerative disease that affects motor neurons, leading to voluntary muscles worsening weakness until death from respiratory failure.49 Variations in more than 40 genes have been found to be associated with the pathogenesis of ALS,50 including the large GGGGCC repeat expansion in C9ORF72 35 and intermediate-length CAG expansion in Ataxin-1 51 and Ataxin-2.37 Moreover, deposition of ubiquitin-positive neuronal intranuclear inclusions was detected both in ALS52 and NIID.3

Yuan and colleagues53 screened 545 patients with ALS and identified four patients harbouring 44, 54, 96 and 143 NOTCH2NLC GGC repeats, claiming that GGC repeat expansion in NOTCH2NLC is associated with ALS (p<0.01). Compared with patients affected by muscle weakness-dominant NIID, patients with NOTCH2NLC-ALS showed a more severe phenotype, more rapid deterioration53 and more frequent upper motor neuron signs. EMG showed spontaneous potentials in four regions of patients with NOTCH2NLC-ALS, which only showed in upper and lower limbs of patients with NIID-M.53 A key question was posed: are the patients with NOTCH2NLC-ALS actually affected by ALS-dominant NIID? Establishing a definitive ALS diagnosis without a relatively long-term follow-up is difficult. According to the NIID diagnostic flowchart,1 the two patients with ALS harbouring 96 and 143 GGC repeats might be diagnosed with ALS-dominant NIID instead of ALS. However, the data of DWI and skin biopsies in the two patients with intermediate-NOTCH2NLC-ALS were not shown, leaving the diagnosis unclear. Given that the patients with intermediate-NOTCH2NLC-PD showed no DWI U-fibre high signal and deposition of ubiquitin-positive and p62-positive intranuclear inclusions, the two patients with intermediate-NOTCH2NLC-ALS might neither radiologically nor pathologically be diagnosed with NIID.

NOTCH2NLC-AD and NOTCH2NLC-FTD

AD and FTD are common neurodegenerative dementias. Dementia is the most prominent symptom (100%) in familial NIID cases with age at onset over 40 years.1 Tian and co-workers4 identified 2/140 families affected by AD harbouring the NOTCH2NLC GGC repeat expansion ranging between 91 and 268. The two pedigrees were eventually recognised as dementia-dominant NIID.4 Jiao and colleagues identified three patients with NOTCH2NLC-AD harbouring 133, 120, 110 GGC repeats (no statistical significance was observed) and one patient with NOTCH2NLC-FTD harbouring 76 GGC repeats. Despite the negative DWI findings in these four patients, the diagnosis of dementia-dominant NIID could not be ruled out as data of skin biopsies, nerve conduction studies and CSF protein elevation, which play a vital role in NIID diagnosis, were not given. In this study, two patients with intermediate-NOTCH2NLC-AD harbouring 45 and 48 repeats were also reported, but the detailed data were not given.

NOTCH2NLC-leukoencephalopathy

MRI showed obvious leukoencephalopathy in 97.4% of sporadic and 76.9% of familial NIID cases.1 Okubo and co-workers33 identified NOTCH2NLC GGC repeat expansion as the most frequent cause of adult leukoencephalopathy in their cohort, followed by NOTCH3 variants. Similarly, the expanded NOTCH2NLC GGC repeats were found rare in European patients with leukoencephalopathy.11 More work is warranted to elucidate the specific phenotype associated with patients affected by NOTCH2NLC-related leukoencephalopathy.

Potential disease-causing mechanisms of expanded GGC repeats in NOTCH2NLC gene

NOTCH2NL is a human-specific gene encoding a protein that enhances Notch signalling. Four paralogous genes (NOTCH2NLA, NOTCH2NLB, NOTCH2NLC and NOTCH2NLR) have been identified, all residing in chromosome 1.54 GGC repeat expansion in the 5′ UTR of NOTCH2NLC has been found to be associated with NIID,4–8 leukoencephalopathy,33 ET,20–23 MSA,27 PD,28 31 ALS53 and OPDM,40 41 most of which are neurodegenerative disorders, suggesting a contributing role of NOTCH2NLC GGC repeat expansion in neurodegeneration. Three possible disease-causing mechanisms may underly the pathogenesis of NOTCH2NLC GGC repeat expansion: loss-of-function, RNA toxicity, and RAN toxicity.

Repeat expansion can lead to loss-of-function of the respective protein. In FMR1, the large (>200) CGG repeats lead to hypermethylation of CGG repeats and adjacent CpG islands and transcriptional silencing of FMR1; however, the methylation level around the large NOTCH2NLC GGC repeat expansion in patients affected by NOTCH2NLC-NIID and NOTCH2NLC-OPDM was low and not different from that in healthy controls.4 41 The GGC repeat expansion did not affect the NOTCH2NLC mRNA level in the blood,4 41 brain5 and fibroblasts6 of patients with NOTCH2NLC-NIID and NOTCH2NLC-OPDM. However, an upregulation of NOTCH2NLC mRNA in muscle samples of patients with NOTCH2NLC-OPDM has been observed. Moreover, there possibly exists an association of intermediate-length GGC repeat expansion with NOTCH2NLC upregulation and autophagy dysfunction phenotype,31 which probably underlies the pathogenesis of intermediate-NOTCH2NLC-related disorders (eg, PD, ET, CBS, PSP).

Yu and co-workers41 reported two asymptomatic individuals (families of patients with NOTCH2NLC-OPDM) harbouring over 400 NOTCH2NLC GGC repeats but not presented any NIID or OPDM clinical manifestations, nor abnormalities in brain and muscle MRI, nerve conduction studies, electromyography, and skin or muscle biopsies, suggesting that individuals harbouring large GGC expansion may be totally but paradoxically asymptomatic. Interestingly, an increased methylation level was observed in the promoter region of NOTCH2NLC in the blood and muscle DNA of asymptomatic individuals compared with patients with NOTCH2NLC-OPDM and healthy controls.41 The NOTCH2NLC mRNA levels in the blood sample of asymptomatic carriers were found significantly lower than that of the healthy controls. However, the NOTCH2NLC mRNA and protein levels in muscle samples of asymptomatic carriers were found unaltered. This implies that the existence of a disease-causing upper limit in the size of NOTCH2NLC GGC repeats, over which may cause hypermethylation in the promoter region of NOTCH2NLC gene and thereby restrict its transcription. Deng et al 55 suggested the disease-causing repeat size might range from 41 to 300.55 However, some exceptional cases were reported, such as a case with muscle weakness-dominant NIID was found harbouring 517 GGC repeats, arguing that there are other factors that together modify the CpG hypermethylation.55

NOTCH2NLC has variable copy numbers. The frequency of 0, 1 and 2 copies of NOTCH2NLC are 0.4%, 6% and 92% in the normal population,56 indicating that NOTCH2NLC may not be essential. Interestingly, a patient with biallelic-NOTCH2NLC-OPDM (harbouring 674 and 217 repeats) showed a milder phenotype than a patient with monoallelic-NOTCH2NLC-OPDM (harbouring 139 repeats).40 Moreover, a patient with familial NIID harbouring a relatively short biallelic GGC repeat expansion in NOTCH2NLC (60 and 76 repeats) showed a dementia-dominant phenotype; however, a detailed description was not given. The homozygous alleles harbouring NOTCH2NLC GGC repeat expansion may not be necessarily inherited from parents both harbouring heterozygous GGC expansion, as the de novo GGC repeat expansion might also present in sporadic case whose parents both harbour wildtype NOTCH2NLC.33 Moreover, the two paralogs of NOTCH2NLC, the NOTCH2NLA and NOTCH2NLB, which are thought to be more important,56 may also compensate for the dysfunction of NOTCH2NLC with expanded GGC repeats.

RNA transcribed from repeat sequences can induce toxicity by sequestering RNA-binding proteins into intranuclear inclusions and compromising their normal function. Abnormal antisense transcripts were found from the beginning or within the expanded GGC repeats in expansion carriers,6 and such expansion could result in the enrichment of differentially expressed genes in neuronal function.6 RNA fluorescent in situ hybridisation showed that NOTCH2NLC mRNA harbouring expanded GGC repeats could form RNA foci in the nuclei of the skin samples from patients with NIID but not from the asymptomatic carriers or the healthy controls.55 The RNA-binding proteins hnRNP A/B and MBNL1 were identified colocalised with p62 in intranuclear inclusions in samples from patients with NOTCH2NLC-OPDM and NIID.41 55 Sam68, which is the early component of CGG aggregates in FXTAS, was also sequestered in RNA foci and found colocalised with p62 in the intranuclear inclusions of patients with NIID .55

Repeat RNA can also induce toxicity by being translated into toxic protein57 in a non-ATG-dependent way called RAN translation. Identification of eosinophilic intranuclear inclusions in the nervous system and multiple visceral organs in GGC expansion carriers suggests that GGC repeats may cause protein-mediated neurodegeneration, resembling repeat expansion in FMR1 and C9ORF72. NOTCH2NLC gene has two transcript isoforms; the GGC expansion locates in the non-coding region of the transcript isoform 1 (NM_001364012.2) and the coding region of the transcript isoform 2 (NM_001364013.2). Thus, two main toxic protein gain-of-function mechanisms were proposed.41 58 Considering that (1) NOTCH2NLC and glycine are found colocalised with p62 in intranuclear inclusions in muscle samples from patients with NOTCH2NLC-OPDM, (2) GGC repeats locate in the coding region in the transcript isoform 2 of NOTCH2NLC and (3) constructed NOTCH2NLC-(GGC)n expressing vectors within transcript isoform 2 reading frame lead to the generation of NOTCH2NLC-polyGlycine (polyG) protein, neurotoxicity and cell death, Yu and co-workers41 suggested that it is possible that the mutant NOTCH2NLC harbouring expanded GGC repeats might be translated into NOTCH2NLC-polyG protein, which contributes to the toxic gain-of-function and formation of the intranuclear inclusions. Interestingly, NOTCH2NLC expression increases considerably with age,4 further suggesting that the increasing expression of NOTCH2NLC alleles harbouring GGC repeat expansion during ageing may contribute to the pathogenesis of neurodegeneration.

The GGC repeat expansion also locates in the non-coding region of the isoform 1 transcript. Interestingly, mass spectrometry revealed that the NOTCH2NLC 5′ UTR of isoform 1 contains a small upstream open reading frame (uORF) that starts from a canonical AUG start codon located 15 nt before the GGC repeats, spans exons 1 and 2, and ends 8 nt before the ATG start codon of the main NOTCH2NLC open reading frame (ORF). The coding protein was called uN2C (called uN2CpolyG when GGC repeats expanded),58 containing a short 5-amino acid N terminus, a variable central glycine stretch and a 38-amino acid C-terminal part. The uN2CpolyG protein was identified in the intranuclear inclusions in NOTCH2NLC-NIID cases but not in European non-NOTCH2NLC-NIID patients, further suggesting that NIID is a heterogeneous syndrome and other NIID-causing mutations remain to be identified. The different GGC repeat size did not alter the translation of the NOTCH2NLC uORF, but the uN2C seemed to be stabilised by the polyG translated by the expanded GGC repeats, leading to its accumulation in patients with NOTCH2NLC-NIID. The uN2C protein is a novel regulator of DNA damage response. The expanded GGC repeats alter the uN2C localisation and DNA repair, but are insufficient to drive overt DNA repair alterations in NIID cases. Nevertheless, the uN2CpolyG protein could be responsible for the typical NIID pathological manifestations in cells and animals.58 The generation of intranuclear inclusions is likely driven by the polyG expansion because the expression of a solo polyG stretch without uN2C sequence also induces the formation of p62-positive cellular inclusions. Therefore, Boivin and colleagues58 suggested that the NOTCH2NLC-related disorders might belong to a novel class of genetic disorders, the polyG diseases. Thus, the expanded GGC repeat expansion in the 5′ UTR of NOTCH2NLC gene was thought to both produce NOTCH2NLC-polyG and uN2C-polyG. Isoform-specific antibodies are required to further identify which form plays a dominant disease-causing role in NOTCH2NLC-related disorders.

Taken together, evidence suggests that the haploinsufficiency of NOTCH2NLC may not be the main disease-causing mechanism. Rather, a toxic gain-of-function mechanism (toxic RNA and/or protein) may primarily drive the pathogenesis of NOTCH2NLC-related disorders.

Conclusions

GGC repeat expansion in the 5′ UTR of NOTCH2NLC has been established as the most common causative factor for NIID. Screening for NOTCH2NLC GGC repeat expansion in neurological disorders and myopathy has widely extended the spectrum of clinical presentation associated with NOTCH2NLC GGC repeat expansion. Although the GGC repeats have been identified in a small group of patients affected by ET, PD, OPDM, MSA, ALS, AD, with or without statistical significance, we do not currently have post-mortem histopathological information from such NOTCH2NLC GGC-positive patients. Therefore, a definitive NIID diagnosis cannot be ruled out for these genetically positive patients that have been clinically diagnosed with ET, PD or other conditions. A longer follow-up may clarify this uncertainty. The repeat size and frequency of interruptions confer a pleiotropic effect on the clinical phenotype. The disease-causing mechanisms underlying expanded GGC repeats in NOTCH2NLC highly resemble the CGG repeat expansion in FMR1.

Finally, given the frequency of NOTCH2NLC GGC repeat expansion in Asian patients, more studies are warranted in this population, to further understand the role of genetic modifiers in the highly variable clinical phenotype of the NOTCH2NLC-related disorders.

Ethics statements

Patient consent for publication

Ethics approval

This study was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University.

References

Supplementary materials

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Footnotes

  • Contributors CS and YX had the idea for the article, YF and CS performed the literature search and data analysis, YF drafted and critically revised the work.

  • Funding This work was supported by the National Natural Science Foundation of China to CS (grant number 81974211, 81771290), National Key R&D Program of China to YX (2017YFA0105000) and the National Natural Science Foundation of China to YX (grant numbers U1904207, 81530037, 91849115).

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.