Assessing the NOTCH2NLC GGC expansion in essential tremor patients from eastern China

We read the recent paper by Sun et al. (2020) which showed that abnormal GGC repeat expansion in the NOTCH2NLC gene with a detection rate of 5.58% was found in a cohort of essential tremor patients from China. Expanded GGC repeats within human-specific NOTCH2NLC was initially identified as the probable pathogenic genetic cause of neuronal intranuclear inclusion disease (NIID), a rare multisystem neurodegenerative disease characterized by eosinophilic intranuclear inclusions existing in the nervous system and multiple visceral organs (Sone et al., 2019). Interestingly, the expanded GGC repeats in NOTCH2NLC were also detected in affected individuals with other neurodegenerative phenotypes, like parkinsonism, Alzheimer’s disease, frontotemporal dementia and multiple system atrophy (Tian et al., 2019; Fang et al., 2020; Jiao et al., 2020). Do abnormal GGC repeat expansions in the NOTCH2NLC gene cause essential tremor, or is essential tremor a subtype of NIID? Recent studies by Chen et al. (2020) and Ng et al. (2020) proposed that essential tremor is an early symptom of NIID. Another question is whether expanded GGC repeats in NOTCH2NLC gene are a common genetic cause of essential tremor pedigrees. However, a recent study from Singapore reported that pathogenic NOTCH2NLC GGC repeat expansions were found in patients with sporadic essential tremor (1.4%), while not in patients with family history (Ng et al., 2020). And the absence of a positive screening result in a European cohort was also noted (Yau et al., 2020). Therefore, to help address these two questions, GGC repeat of NOTCH2NLC gene as well as the image data were both detected in a cohort of 228 essential tremor probands from eastern China. In the study, a total of 228 probands (from 228 essential tremor pedigrees) from eastern China were included. The patients were recruited at the Department of Neurology, Second Affiliated Hospital, Zhejiang University School of Medicine. The diagnosis of essential tremor was made according to the guidelines of the Consensus Statement of the Movement Disorders Society (Deuschl et al., 1998). They were subjected to a thorough neurological examination by at least two experienced neurologists. Written informed consent was obtained from all study subjects, and the research was approved by the local ethics committees. Repeat-primed PCR (RP-PCR) was conducted to amplify the repeat sequence and GC-rich PCR (GC-PCR) was used to determine repeat size. For GC-PCR and RP-PCR, the primers were used as described previously (Sun et al., 2020). The experimental procedures are available on request. The PCR products were subjected to capillary electrophoresis using the 3730XL Genetic Analyzer for Human Identification (Applied Biosystems) and the data were analysed using GeneMarker software. Allele sizes were determined using GeneScan 1200 LIZ Size Standard (Applied Biosystems). GGC repeats were determined directly by the size of PCR product as determined by ABI 3730XL and compared with the PCR products of known FMR1 (MIM: 309550) premutation CGG repeat alleles of various sizes. Of the 228 probands, 103 were male and 125 were female. The average age at onset of the probands was 45.36 ± 16.66 years, while the mean disease duration was 13.14 ± 12.89 years. The average Tremor Essential Tremor Rating Assessment Scale (TETRAS) score is shown in Supplementary Table 1. The ratio of pedigrees with GGC repeat expansion to all pedigrees was lower than the patients in Sun et al.’s study. RP-PCR and GC-PCR were used to determine the GGC repeat size of the 228 affected members from 228 pedigrees. Expansion of GGC repeats (>60) in NOTCH2NLC was detected in three families (Fig. 1A–C), which showed similar peak patterns using RP-PCR (Fig. 1D). We then used GC-PCR to determine the repeat number and found that the affected individuals from the three families carried alleles ranging from 83 to 102 (Fig. 1E). All of the other patients with no expansion, from 225 pedigrees, carried normal repeats, ranging from 10 to 48, with an average of 22.25 ± 5.63 repeats (Fig. 1F). Representative electropherogram and MRI image of three essential tremor pedigree probands with expanded GGC repeats. (A–C) Pedigrees of Families 1–3 are shown. Filled symbols indicate affected individuals; open symbols indicate unaffected members. (D and E) Validation of GGC repeat expansion and repeat size determination by RP-PCR and GC-PCR assays. RP-PCR (D) and GC-PCR assays (E) of one patient showed abnormal repeat expansions. (F) GGC repeat sizes among 225 patients ranged from 10 to 48, with an average of 22.25 ± 5.63 repeats. (G) Brain MRI of one essential tremor pedigree proband with expanded GGC repeats (Family 3, Patient III:2). Some ribbon-like high-intensity signals along the corticomedullary junction on diffusion weighted imaging in the patient could be seen, although not obvious (white arrowhead). Representative electropherogram and MRI image of three essential tremor pedigree probands with expanded GGC repeats. (A–C) Pedigrees of Families 1–3 are shown. Filled symbols indicate affected individuals; open symbols indicate unaffected members. (D and E) Validation of GGC repeat expansion and repeat size determination by RP-PCR and GC-PCR assays. RP-PCR (D) and GC-PCR assays (E) of one patient showed abnormal repeat expansions. (F) GGC repeat sizes among 225 patients ranged from 10 to 48, with an average of 22.25 ± 5.63 repeats. (G) Brain MRI of one essential tremor pedigree proband with expanded GGC repeats (Family 3, Patient III:2). Some ribbon-like high-intensity signals along the corticomedullary junction on diffusion weighted imaging in the patient could be seen, although not obvious (white arrowhead). The main clinical manifestations of the three expansion (+) patients are shown in Table 1. Because several family members had a similar pattern of tremor with more than 10 years of disease duration, the diagnosis of familial essential tremor was made. Interestingly, brain MRI of one proband (Family 3, Patient III:2) showed suspicious abnormal diffusion weighted imaging signals in Fig. 1G. Therefore, NIID was also suspected. However, all the patients with expanded GGC repeats were unwilling to take skin biopsy. Summary of the clinical features of the three probands DWI = diffusion weighted imaging; MMSE = Mini-Mental State Examination; TETRAS = Tremor Essential Tremor Rating Assessment Scale. Summary of the clinical features of the three probands DWI = diffusion weighted imaging; MMSE = Mini-Mental State Examination; TETRAS = Tremor Essential Tremor Rating Assessment Scale. In this study, we found three families with abnormal GGC repeat expansion in the 5′ region of the NOTCH2NLC gene in 228 essential tremor pedigrees. This result was consistent with the findings in studies from Asia, but not with that of Europe (Ng et al., 2020; Sun et al., 2020; Yau et al., 2020). Since our cohort is mainly from eastern China, further studies involving populations from different ethnic origins and regions are needed to further establish the relevance and contribution of NOTCH2NLC to the pathogenesis of essential tremor. Unlike Parkinson’s disease, the exact pathogenesis of essential tremor remains elusive. Several possible underlying mechanisms include GABA-ergic dysfunction, cerebellar neurodegeneration, and olivary dysfunction (Hopfner and Helmich, 2018). Sun et al. (2020) found that skin biopsies for the three patients with expanded GGC repeats were positive and another one with normal GGC repeats was negative (Sun et al., 2020). As eosinophilic intranuclear inclusions are characteristic of NIID, we proposed that, although the three patients were clinically diagnosed as having essential tremor, they may be diagnosed as NIID after a long follow-up with essential tremor as a special phenotype of NIID (Westenberger and Klein, 2020). This view was supported by Chen et al. (2020), who provided MRI evidence for a case with expanded GGC repeat and pathological changes. In our study, one case with suspicious zigzag edging signs along the corticomedullary junction on diffusion weighted imaging was indicated. Follow-up study and skin biopsy would provide more evidence to explain the relationship between essential tremor and NIID. In summary, our study has provided further evidence for the support of the role of GGC repeat expansions in the 5′ region of the NOTCH2NLC gene in Chinese patients with an essential tremor phenotype. NOTCH2NLC gene test and skin biopsies are suggested in the early diagnosis of NIID patients. The exact mode of inheritance and pathogenicity of essential tremor patients with positive family history still requires more genetic studies in large essential tremor pedigrees. The data that support the findings of this study are available from the corresponding author, upon reasonable request. This work was supported by grants from the National Natural Science Foundation of China (No. 81570698, 81520108010 and 81200984), the Natural Science Foundation of Zhejiang Province (LY16H070003), the National Basic Research Development Program of China (No.2013CB530900 and 2013CB530904), the General Medical and Health Project of Zhejiang Province (No. 2018RC004, 2019DZ021 and 2020RC061). The authors report no competing interests. Supplementary material is available at Brain online.