Abstract
Background
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease with neuronal cell inclusions composed of neurofilaments and other abnormal aggregative proteins as pathological hallmarks. Approximately 90% of patients have sporadic cases (sALS), and at least 4 genes, i.e. C9orf72, SOD1, FUS and TARDBP, have been identified as the main causative genes, while many others have been proposed as potential risk genes. However, these mutations could explain only ~ 10% of sALS cases. The neurofilament polypeptides encoded by NEFH, NEFM, and NEFL are promising protein biomarkers for ALS and other degenerative diseases. However, whether the genetic variants of these genes were associated with ALS remain ambiguous.
Methods
Here, we used PCR-Sanger to sequence the exons of these three genes in a cohort of 371 sALS patients and 711 healthy controls (Phase I) and validated the risk variant in another 300 sALS patients and 1076 controls (Phase II).
Results
A total of 92 variants were identified, including 36 rare heterozygous variants in NEFH, 27 in NEFM, and 16 in NEFL, and only rs568759161 (p.Ser787Arg) in NEFH reached nominal statistical power (P = 0.02 at Phase I, P = 0.009 at Phase II) in the case–control comparison. Together, the Phase I and II studies showed the significantly higher frequency of the variant in cases (9/1342, 0.67%) than in controls (2/3574, 0.07%) (OR 12.06; 95% CI 2.60–55.88; P = 0.0003). No variants passed multiple testing in the discovery cohort, but rs568759161 was associated with ALS in a replication cohort.
Conclusions
Our results confirmed that NEFH Ser787Arg is a novel sALS risk variant in Chinese subjects, but NEFM and NEFL were not associated with sALS. These data may have implications for genetic counselling and for understanding the pathogenesis of sALS.
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Introduction
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by loss of motor neurons in the brain and spinal cord, resulting in muscle atrophy, swallowing disorders, and pyramidal tract signs. A known pathological hallmark of ALS is neuronal cell inclusions composed of neurofilaments and other abnormal aggregative proteins [1,2,3]. Epidemiological surveys show an incidence of 0.6–3.8 per 100,000 persons per year and a prevalence of 4.1–8.4 per 100,000 persons worldwide [4]. It has been reported that the yearly incidence is 0.8 (2010–2015) per 100,000 persons in China [5]. Approximately 10% of cases were familial, and 90% were sporadic cases. To date, the genetics of ALS are not fully understood. In 1993, SOD1 was discovered as the first ALS-causing gene. Since then, many other genes have been reported to be causative for (i.e. C9orf72, SOD1, FUS, TARDBP, etc.) or associated with the disease [6, 7]. Genetic studies have found that mutations in these genes were mainly identified in familial cases and could explain only approximately 10% of sporadic cases (sALS) [6]. With next-generation sequencing, novel genes and loci have been increasingly discovered [40], and motor neuron disease in mice [32]. NEFM is linked to Parkinson's disease [41]. However, NEFL and NEFM were not associated with sALS in our study. Although we had shown variants in these genes in sALS, we did not find significant differences in clinical characteristics (sex, AAO, onset site) between cases carrying and not carrying the variants. The differences further confirmed the genetic heterogeneity in sALS among different ethnicities and highlight the association of NEFH, but not NEFL or NEFM, with ALS.
This study found that the p.Ser787Arg variant in NEFH was associated with sALS in Chinese subjects. Notably, rs568759161 is only found in only the East Asian population according to ExAC (MAF 0.14%) and gnomAD (MAF 0.24%), and their MAFs were slightly higher than those of our control group (0.07% in Phase I and 0.05% in Phase II) (Table 3). We assume that the difference in allele frequency between the two databases and our study might be due to the population differences. Moreover, we found that some variants reported by other studies were not associated with ALS. For example, A380T in NEFH was identified only in cases previously [29], but our study suggested it was identified in both case and control groups. So, we believed study of rare variants need large samples of controls. In our study, we recruited relatively large controls (n = 711 in Phase I and n = 1076 in Phase II) to decrease the chance of false positive or false negative.
The phosphorylation of NF subunits has been considered a critical process regulating the formation and function of NFs [10]. The variant p.Ser787Arg is located in the phosphorylated region in a conserved sequence. Proper phosphorylation/dephosphorylation of NEFH may be considered a protective mechanism under conditions of cellular stress [16, 42], indicating that the modification of NEFH plays a significant role in maintaining the normal function of neurons. We hypothesized that the NEFH-S787R variant changes the phosphorylation of the protein. However, because of the unavailability of an antibody against the site, we did not test the hypothesis in this study. In the future, we need to synthesize antibodies against the phosphorylated NEFH-Ser787 site to further explore the changes in phosphorylation levels.
Recently, next-generation sequencing technology have identified many genes, including NEFH [43], as causative for or associated with ALS. In NEFH, 20 variants have been reported in ALS cases (Fig. 1) [25, 36,37,38, 44, 45], but none was conclusively related to the disease. The p.Ser787Arg was only reported by Chen et al. [45], but its association with ALS was not confirmed. Our study provided the spectrum of NEFH variants and confirmed the association of p. Ser787Arg with Chinese sALS.
Conclusion
In this study, we analysed the mutational spectrum of NEFH, NEFM, and NEFL genes in an sALS Chinese cohort and identified the variant (rs568759161) locating in the phosphorylated site of the KSP domain of NEFH as a risk variant associated with sALS in Chinese. Functional studies will be necessary to assess its role in ALS pathogenesis.
Availability of data and materials
All genetic polymorphisms identified in this study is available at Table 2, Additional file 2: Table S2 and Additional file 5: Table S5. The primer sequences are shown in Additional file 1: Table S1. The original sequencing and clinical datasets generated during the current study are not publicly available due to maintaining patient confidentiality but are available from the corresponding author (hh-p@163.com) on reasonable request.
Abbreviations
- ALS:
-
Amyotrophic lateral sclerosis
- NFs:
-
Neurofilaments
- FTD:
-
Frontotemporal dementia
- AAO:
-
Age at onset
- PCR:
-
Polymerase chain reaction
- ExAC:
-
Exome aggregation consortium
- MAFs:
-
Minor allele frequencies
- CADD:
-
Combined annotation-dependent depletion
- SKAT-O:
-
Sequence kernel association test
- LoF:
-
Loss-of-function
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Acknowledgements
We thank all the participants of this study. We’re also grateful for Dr. **uli Feng and Dr. Shu **e for their technical assistance, including PRC and Sanger sequencing.
Funding
This study was supported by funds from the Innovation of Science and Technology, Fujian Province (Grant Number 2017Y9058), to Prof. Huapin Huang; the Fujian Provincial Health Technology Project (Grant Number 2019-ZQN-38), to Dr. Wanhui Lin; the Fujian Sanming Science and Technology Plan Project (Grant Number 2019-S-3) and Fujian Provincial Science and Technology Project (Grant Number 2020J011271), to Dr. Feng Lin; and the Ministry of Science and Technology (Grant Number 2016YFC1306000), Special Fund from Key Laboratory of Neurodegenerative Diseases, Ministry of Education of China (PXM2019_026283_000002), and National Natural Science Foundation (Grant Number 81771212) to Prof. Chaodong Wang.
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FL and HH initiated the project and wrote the manuscript. FL, WL, and CZ designed the experiments. FL, CZ, JL, and XL performed the experiments. FL, ZW, and CW analysed the data. FL, XL, JZ and JL constructed the figures. FL, XL, JZ and XL created the tables. All authors read and approved the final manuscript.
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All research participants or their legal representatives signed informed consent forms for participation in clinical and genetic research. The medical ethics committee approved the protocol and provided informed consent (2021KY016) of the whole study at Fujian Medical University Union Hospital, Fuzhou, China. In this study, all methods were performed in accordance with the relevant guidelines and regulations in accordance with the Declaration of Helsinki.
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Supplementary Information
Additional file 1
. Primers for amplification of exons.
Additional file 2
. Rare non-synonymous coding variants in NEFM and NEFL.
Additional file 3
. Burden in all rare variants.
Additional file 4
. Clinical features of NEFH (S787R) carriers.
Additional file 5
. Low frequency and common variants identified in NEFH, NEFM and NEFL genes.
Additional file 6
. Workflow of the study design.
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Lin, F., Lin, W., Zhu, C. et al. Sequencing of neurofilament genes identified NEFH Ser787Arg as a novel risk variant of sporadic amyotrophic lateral sclerosis in Chinese subjects. BMC Med Genomics 14, 222 (2021). https://doi.org/10.1186/s12920-021-01073-z
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DOI: https://doi.org/10.1186/s12920-021-01073-z