FUS P525L mutation causing amyotrophic lateral sclerosis and movement disorders

Abstract Background Mutations in the fused in sarcoma (FUS) gene have been associated with amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration, and essential tremor. Among the FUS mutations, p.P525L as a hot spot variant has been reported in more than 20 patients with ALS. Apart from the typical ALS phenotype, patients with p.P525L mutation exhibit some atypical symptoms. However, movement disorders related to p.P525L mutation have not been emphasized currently. Methods Two unrelated patients with ALS were evaluated through a set of clinical and laboratory tests. The genetic screening was performed through next‐generation sequencing. Muscle biopsies were performed on the 2 patients. Muscle samples were stained according to standard histological and immunohistochemical procedures. Results The first patient presented with juvenile‐onset neurogenic weakness and wasting and simultaneously had dropped head, ophthalmoplegia, tremor, involuntary movements, and cognitive impairments. The second patient showed a typical ALS phenotype and prominent adventitious movements. Genetic screening disclosed de novo p.P525L FUS mutation in the 2 patients by family cosegregation analysis. Muscle biopsy showed neurogenic patterns and numerous lipid droplets aggregating in the fibers. Conclusion Apart from the typical ALS phenotype, patients with p.P525L mutation in the FUS gene can present with great clinical heterogeneity including multiple movement disorders. Numerous lipid droplets in muscle fibers indicate that skeletal muscle is likely an important therapeutic target for ALS.

Typical symptoms of ALS present with weakness and wasting of the limbs, bulbar paralysis, and respiratory insufficiency. Approximately 90% of patients are sporadic ALS (SALS), and the remaining 10% of patients are familial ALS (FALS) (Chia, Chiò, & Traynor, 2018). Most patients with ALS have adult onset of age with a peak age at onset of 47-52 years for FALS and 58-63 years for SALS (Kiernan et al., 2011).
To date, there are nearly 30 genes that are responsible for genetic causes of ALS or are highly correlated with ALS. Since two academic groups simultaneously identify the fused in sarcoma (FUS) gene as one of genetic causes of ALS (Kwiatkowski et al., 2009;Vance et al., 2009), more than 100 FUS mutations have been recorded in the Human Gene Mutation Database (Shang & Huang, 2016). FUS is an RNA-binding protein whose genetic mutations or pathological inclusions are currently associated with multiple neurological diseases including ALS (Vance et al., 2009), frontotemporal lobar degeneration (FTLD;Cairns & Ghoshal, 2010), and essential tremor (ET; Merner et al., 2012), although the results of ET in most confirmatory studies are negative (Zheng et al., 2013). A meta-analysis including 154 ALS cases with FUS mutations shows an average disease onset of 43.8 ± 17.4 years. More than 60% of cases with FUS mutations show disease onset younger than 45 years of age, evenly many juvenile-onset ALS cases present with disease course in their late teens or early 20s (Huang et al., 2010).
Most patients with FUS mutations show a typical ALS phenotype, but some patients display a variety of accompanying symptoms including parkinsonism-like symptoms (Yan et al., 2010), myoclonic jerks (Dodd, Power, Ealing, & Hamdalla, 2019), and peripheral neuropathy (Mackenzie et al., 2011). Among the FUS mutations, p.P525L mutation as a hot spot variant has been reported in more than 20 patients with ALS. Besides the typical symptoms, the phenotype associated with p.P525L mutation concomitantly exhibits some atypical symptoms such as developmental delay (Mochizuki et al., 2012), ophthalmoplegia (Leblond et al., 2016), cognitive impairments (Yan et al., 2010), and tremor (Eura et al., 2019). In this study, we describe two patients with FUS p.P525L mutation presenting with rapid progressive ALS and multiple movement disorders, which indicate the great clinical heterogeneity associated with FUS p.P525L mutation.

| Subjects
Patients were recruited at the neurological department of the first affiliated hospital of Nanchang University from January 2018 to December 2019. ALS was diagnosed on a clinical and electrophysiological basis of the revised El Escorial criteria. Initial symptoms, age of onset, progression of disability, and clinical manifestations were recorded as reported by the patients and their relatives. Time of disease duration was calculated from the reported date of symptom onset to the initial medical consultation in our department. All samples and medical data were obtained after a written consent signed by each individual in compliance with the bioethical laws of China as well as the Declaration of Helsinki.
The research was also approved by the ethics committee of the first affiliated hospital of Nanchang University.

| Genetic testing
Genomic DNA was extracted from peripheral blood samples.
Targeted exon enrichment was performed using SureSelect Human All Exon V5 (Agilent Technologies). The exon-enriched DNA libraries were subjected to paired-end sequencing with the Hiseq2000 platform (Illumina, Inc.). Sequence data were mapped with BWA and SAMTOOLS onto the hg38 human genome as a reference. Calls with variant quality less than 20 were filtered out, and 95% of the targeted bases were covered sufficiently to pass our thresholds for calling single nucleotide polymorphisms (SNP), nonsynonymous/splice acceptor and donor site, insertions or deletions (NS/SS/InDel) variants in the dbSNP v137, ESP6500, and 1,000 Genome were removed.
Synonymous changes were filtered using SIFT software (http://sift. jcvi.org). Sanger sequencing with specific primers was conducted to confirm the FUS mutation in the patients and their family members (parents and siblings). Analysis of the hexanucleotide repeats in C9orf72 and the trinucleotide repeats in the Huntingtin (HTT) gene was performed by a repeat-primed polymerase chain reaction (RP-PCR).

| Muscle pathological examination
Muscle biopsies were performed from the left bicep of the two cases.
The muscle tissue was frozen and then cut at 8-μm sections. These

| Case 1
A 19-year-old young man was referred to our department with a history of cervicodorsal pain and dropped head for 3 months The results of a nerve conduction study were normal, but needle electromyography showed acute and chronic denervation in four segments.

| Case 2
A 34-year-old man was referred to our department with progressive limb weakness for 6 months, adventitious movements for 5 months, and dysphagia for 2 months (Figure 1b). The man had no family history of other neurological diseases including muscle weakness and extrapyramidal disorders. Physical examination on admission revealed that an emaciated man presented with adventitious movements characterized by eyebrows squeezing, neck torsion, torso, and limb twisting. The movements involved all body parts including the face, tongue, neck, torso, and limbs. These movements were repetitive, but not rhythmic, stereotyped, or high speed, flowing from side to side, from limb to limb, and merging into the appearance of continuous motion (Video S2). The On the basis of these findings, we administered case 1 for riboflavin (50 mg tid) and levocarnitine (1 g, bid). The patient can partially lift his head one week later (Video S3), but no further benefits were observed one month later.
As for case 2, the number of CAG repeats of HTT was initially identified with 16 and 19 copies at the two allelic chromosomes, respectively. A whole-genome sequencing was then performed and  (Table S2).

| D ISCUSS I ON
The clinical classification of ALS associated with FUS mutation has been designated as familial ALS type 6 (ALS6; Vance et al., 2009 neuron with young onset, aggressive course, high incidence of bulbar symptoms, and early respiratory involvement, although typical ALS phenotype and slower disease course have also been described in some patients (Naumann et al., 2020;Sproviero et al., 2012). FUS mutations have been reported to be responsible for 3%-4% of familial ALS and for less than 1% of sporadic ALS (Zou et al., 2013).
Among these mutations, p.P525L FUS mutation has been reported to be consistently associated with younger onset, rapid disease course, and high proportion of de novo mutations in sporadic patients (Conte et al., 2012). We summarized the demographic data and clinical features of all reported patients with p.P525L/R mutation (Table 1; (Koutsis, Karadima, Kartanou, Kladi, & Panas, 2015).
FUS is one of the major components of nuclear polyQ aggregate-interacting proteins in Huntington's disease and was also associated with neuronal intranuclear inclusions (Kino et al., 2016).
Because FUS has a variety of functional roles, the aggregating FUS may play a role in diverse pathological changes in the brains of patients with ALS6 and induce neurodegeneration of multiple systems including the frontal lobe, the basal ganglia, substantia nigra, cerebellum, and other related areas (Mochizuki et al., 2012). Compared with previously reported ALS6 cases, the prominent movement disorders were the characteristic features in both patients, indicating that FUS mutation can affect the function of movement coordinating system in addition to motor neuron degeneration. It is open to discuss if this is a coincidental finding or linked to the identified mutation. Given the oligogenic nature of ALS, we also identified several variants associated with ALS-FTD, Parkinson's disease, and Alzheimer's disease through exome sequencing. These variants might act as disease modifiers, whereas the variants were benign or uncertain significance, and the oligogenic relationship would be difficult to be clearly evaluated.
One very interesting clinical feature observed in patient with FUS mutation was a severely disabling dropped-head syndrome, which emerged as the initial symptom in our first patient. Primary droppedhead syndrome is usually caused by cervical extensor myopathy of unknown etiology, while secondary dropped-head syndrome is mostly associated with motor neuron disease (Peng et al., 2015). Our patient with p.P525L mutation showed a neck pain and a significant decrease of cervical extensor muscle strength, which have not been reported in previous cases. Patients with mutations at other FUS loci (e.g., at position 521) also showed axial muscle atrophy and profound weakness as previously reported (Blair et al., 2010;Naumann et al., 2020). So this phenotype is likely to be characteristic of individuals with C-terminal FUS mutations.
The muscle biopsy specimens of the two patients showed acute or subacute neurogenic processes with angular fibers grouping and target-like fibers, which were consistent with the results of previous studies (King et al., 2015). Eura et al. had observed the muscle pathology in an ALS patient with p.P525L FUS mutation and found no abnormal inclusions or aggregations (Eura et al., 2019). We neither observed aggregation of FUS inclusions in the two patients with p.P525L FUS mutation, suggesting p.P525L mutation directly unaffected muscle pathology. ALS patients' or models' muscles exhibited increased reliance on fatty acids in physiological studies, while the extent of lipid storage in ALS muscles was rarely elucidated (Palamiuc et al., 2015). To our surprise, we found that numerous lipid droplets were deposited in the relatively hypertrophy fibers.
Therefore, the patient 1 was given riboflavin and levocarnitine to modify the lipid metabolism and got a partial improvement. Animal model with p.P525L mutation exhibited that mutant FUS disrupted assembly and function of the mitochondrial ATP synthase complex and possibly resulted in irreparable mitochondrial damage that might cause impediment of lipid metabolism pathway (Deng et al., 2018).
We provided evidence that muscle metabolic alterations occurred in motor neuron degeneration, indicating that skeletal muscle was likely an important therapeutic target for ALS.
Full-length human FUS protein can be divided into Q/G/S/Y domain, G-rich region, RNA recognition motif (RRM), two Arg-Gly-Gly (RGG)-repeat regions interrupted by a zinc finger motif (ZNF), and nuclear localization signal (NLS) (Shang & Huang, 2016). Structurefunction analyses have shown that the G-rich domain (amino acids 156-262) and C-terminal domain (amino acids 450-526) of FUS are required for interaction of FUS and histone deacetylase 1 (HDAC1) (Shang & Huang, 2016), which harbor most of the ALS mutations ( Figure 3c). The high frequency of de novo p.P525L mutation may be associated with the high GC content in the NLS domain, also be associated with a reduced life expectancy or reduced reproductive fitness. Overall, p.P525L mutation leads to a stronger cytosolic mislocalization of FUS protein, consistent with the phenotype of an earlier age of onset and a more aggressive disease process in the patients with the mutation (Ito et al., 2011).
In conclusion, FUS mutations may affect a broader range of functions in addition to degeneration of motor neurons. Apart from the typical ALS phenotype, patients with p.P525L mutation in the FUS gene can present with great clinical heterogeneity including tremor, movement disorders, dropped-head syndrome, and cognitive impairments. Numerous lipid droplets in muscle fibers indicate that skeletal muscle is a possible therapeutic target for ALS.

ACK N OWLED G M ENTS
We thank the patients and their families for cooperation. We also thank Ms. Ling Liu for pathological technician.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N
ZB, WH, WH, and WL contributed to the acquisition and analysis of data. ZM, CY, and YY performed the genetic analysis. LX, ZM, and LX performed the pathological study. FP and CY contributed to critical revision of the manuscript. ZB and HD contributed to the study design and drafted the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All relevant data are within the paper and its Supporting Information files.