Introduction
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by aggressive degeneration of upper and lower motor neurons. Patients usually present with muscular weakness and atrophy, and they die from respiratory failure three to four years after the disease onset.[1] Juvenile amyotrophic lateral sclerosis (JALS) is an uncommon form of ALS whose age at onset (AAO) is defined as prior to 25 years with phenotypic variability.[2] It is universally known that genetics play significant roles in the pathogenesis of JALS. Over the last 30 years, more than ten causative genes have been identified in JALS.[3]
The SPTLC1 gene encodes the long-chain base subunit one of serine palmitoyltransferase (SPT), which is the critical rate-limiting enzyme in the initial step of sphingolipid biosynthesis.[4] In 2001, SPTLC1 was identified as the causative gene for hereditary sensory neuropathy type 1 (HSAN1),[5] and mutations identified in patients with HSAN1 reduced SPT activity, leading to the accumulation of pathogenic and neurotoxic sphingolipids.[6] Recently, SPTLC1 was identified as a disease-causative gene for JALS, and its mutations promoted enzymic activity, resulting in an increase in standard products.[7,8] To date, a novel SPTLC1 (c.113T>G, p.L38R) mutation has been identified in a Chinese JALS patient,[9] but other SPTLC1 mutations have not been reported in Chinese patients with JALS.
The FUS gene contains 15 exons and encodes a protein consisting of 526 amino acids. Mutations in FUS were described as the most common cause of the autosomal dominant form in JALS, and the majority was de novo.[10,11] Notably, mutations in SPTLC1 occurred de novo in ten out of 16 JALS patients carrying SPTLC1 mutations,[7-9] indicating de novo mutagenesis as the underlying genetic mechanism. However, little is known regarding the difference in clinical features between JALS patients carrying FUS and SPTLC1 mutations. Therefore, in this study, we screened SPTLC1 and FUS mutations in JALS patients and compared the corresponding clinical features to better understand the genotype–phenotype correlation of JALS.
Methods
Ethical approval
This study was approved by the Ethics Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine (No. 2015-048). Written informed consent was signed by participants or their legal guardians.
Patients and sample collection
A total of 16 JALS patients were included in this study. Three of them were newly recruited between July 2015 and August 2018 from the Second Affiliated Hospital, Zhejiang University School of Medicine, and the other 13 were reported in our previous studies.[11,12] All patients were evaluated by two experienced neurologists, underwent electromyography (EMG), and fulfilled the revised El Escorial criteria.[13] Blood samples from available family members were also obtained.
DNA extraction, sequencing, and analysis
Genomic DNA was extracted from peripheral white blood cells using QIAamp Blood Genomic Extraction Kits (Qiagen, Hilden, Germany) following the standard instructions. Whole-exome sequencing (WES) was performed using an Agilent SureSelect Human All Exome V6 kit (Agilent Technologies Inc., Santa Clara, CA, USA). Then, the captured 150-bp library fragments were sequenced on an Illumina HiSeq X Analyzer (XY Biotechnology Co. Ltd., Hangzhou, China) and annotated. Public databases, including the Genome Aggregation Database (gnomAD), Exome Aggregation Consortium (ExAC) database, National Heart, Lung, and Blood Institute Grand Opportunity (NHLBI GO) Exome Sequencing Project (ESP6500), and 1000 Genomes Project (1000G) database, were used to estimate the frequency of the identified variants in the general population. In silico prediction of functional effects was evaluated using Sorting Intolerant from Tolerant (SIFT, https://sift.bii.a-star.edu.sg/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), Combined Annotation Dependent Depletion (CADD, https://cadd.gs.washington.edu/), and Mutation Taster (http://www.mutationtaster.org/). The National Center for Biotechnology Information Web (NCBI, https://www.ncbi.nlm.nih.gov) was used to evaluate the evolutionary conservation of mutant amino acids. Sanger sequencing was carried out to validate the variant and co-segregation in each proband and available familial members. Haplotype analysis was performed with GlobalFiler PCR amplification kits according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA).
Literature review
We reviewed all of the research considering JALS patients carrying SPTLC1 and FUS mutations through PubMed based on the following terms: JALS and specific genes (SPTLC1 and FUS). After screening all articles or case reports in English, 32 articles were retrieved (including a preprint of a novel SPTLC1 mutation identified in a Chinese JALS patient). Inclusion criteria were defined as having both genetic information (FUS or SPTLC1 mutation) and phenotypic descriptions (including AAO, onset site, and disease duration). Cases met the inclusion criteria were included for further analyses.
Statistical analyses
Statistical analyses were performed using SPSS version 26.0 (IBM, Armonk, NY, USA). Categorical variables were displayed as numbers of cases (%). Continuous variables with normal distribution were shown as mean ± standard deviation; non-normally distributed variables were presented as median (interquartile range). Clinical features were compared between the two groups using Student's t test for AAO and the χ2 test for gender and onset site. Clinical features were compared among the three groups using one-way analysis of variance (ANOVA) followed by the Scheffe post hoc test with different sample sizes for AAO and the χ2 test followed by a Bonferroni post hoc test for gender and onset site. Fisher's exact test was used when the expected frequency in one or more cells was less than five. Kaplan–Meier analysis was performed to compare survival between groups. Statistical significance was defined as a P value <0.05.
Results
Genetic findings and mutation spectrum in our JALS patients
WES was performed on the three newly recruited JALS patients. A novel variant (NM_006415.4: c.58G>A, p.A20T) within SPTLC1 was identified in a sporadic JALS patient (patient 1, II-1 in Figure 1A) after WES analysis and Sanger sequencing, which was absent in his parents. Then, we performed haplotype analysis and confirmed that the variant was de novo [Figures 1A and 1B]. This variant was not observed in gnomAD, ExAC, ESP6500 and the 1000G database, nor in our in-house dataset comprising 1000 unrelated Chinese individuals. In silico prediction of this variant revealed an extremely deleterious possibility (Polyphen-2=0.999, REVEL=0.529, ClinPred=0.946, and CADD=25.3) and high evolutionary conservation among multiple species [Figure 1C]. The location of this variant was in exon 2, which differed from those mutations detected in HSAN1 patients [Figure 1D]. By using University of California, San Francisco Chimera version 1.15 (University of California, San Francisco, CA, USA), we found that the position of the 20th amino acid in SPTLC1 was close to ORMDL3 in the 3D protein structure [Figure 1E]. Based on the American College of Medical Genetics and Genomics Guideline (ACMG), the variant was classified as “pathogenic” (PS2 + PM1 + PM2 + PM5 + PP3). Moreover, we identified one known FUS mutation (NM_004960.4: c.1574C>T, p.P525L) in patient 2 [II-1 in Figure 1F], which was genetically confirmed as a de novo mutation by Sanger sequencing and haplotype analysis of parental DNA [Supplementary Table 1, https://links.lww.com/CM9/B339]. Therefore, as shown in Figure 1G, in our 16 JALS patients, 7/16 patients carried FUS mutations, including three with p.P525L mutation and four with different frameshift mutations. No pathogenic mutation in known ALS-related genes was detected in another 4/16 patients.
;)
Figure 1: Genetic analysis in JALS patients and their clinical features. (A) Pedigree of Family 1 and haplotype analysis. Square indicates male; circle indicates female; solid symbol indicates affected individual, and arrow indicates the proband. (B) Sequence chromatograms of SPTLC1 mutation. The red arrow and red frame indicate the mutation site. (C) Amino acid conservation of the SPTLC1 mutation in multiple species. The red frame indicates the 20th amino acid in SPTLC1. (D) Distribution of SPTLC1 mutations (NM_006415.4) in JALS and HSAN1 patients. Mutations in HSAN1 are shown in blue; those in JALS are shown in black; and those in both HSAN1 and JALS are shown in green. The novel one is in red. (E) Three-dimensional structure of the human SPT complex: SPTLC1/SPLTC2/ssSPTa/ORMDL3. The red arrow indicates the position of the 20th amino acid in SPTLC1, which is close to ORMDL3. (F) Pedigree of Family 2. Square indicates male; circle indicates female; solid symbol indicates affected individual; and arrow indicates the proband. (G) Genetic spectrum in our JALS cohort. FUS mutations were identified in seven patients (43.8%), and mutations in SPTLC1, SETX, NEFH, DCTN1, and TARDBP were identified in one patient each. (H) X-ray film of the chest showed severe scoliosis in patient 1. (I) Photograph of the lower extremities from patient 1. The photo was taken at the age of 13 years in the last review and showed atrophy of the lower extremities and pes cavus. HSAN1: Hereditary sensory neuropathy type 1; JALS: Juvenile amyotrophic lateral sclerosis; SPT: Serine palmitoyltransferase.
Clinical features of patients
Patient 1 carried the novel and de novo mutation within SPTLC1 (c.58G>A, p.A20T) and was a boy with lower limb onset. When he was 7 years old, his parents noticed that he experienced toe walking. He could not run as fast as other children of the same age and was prone to falling down. Soon after, he developed walking difficulty and muscle weakness in his lower limbs. Three years later, the weakness gradually progressed to the bulbar muscles, and he had some trouble in drinking water and swallowing food. Neither sensory nor cognitive impairment was observed. Neurological examinations at the age of 10 years showed marked atrophy of the tongue as well as weakness of neck muscle (Muscle Strength Grading Scale 2/5). His muscle strength decreased remarkably in distal areas of the lower limbs (0/5) compared with other parts (4/5). The extremities’ deep tendon reflexes were brisk. Hoffman's sign, ankle clonus, and Babinski sign were positive. EMG at that time revealed a reduced compound muscle action potential (CMAP) of the right median (2.8 mV) and peroneal motor nerves (0.8 mV). Nerve conduction velocities and sensory nerve action potential (SNAP) were normal [Supplementary Table 2, https://links.lww.com/CM9/B339]. Fibrillation potential, sharp waves, and prolonged polyphasic motor unit potentials were observed in the right first dorsal interosseous muscle, rectus abdominis, and tibialis anterior muscle. During a 9-year follow-up period, weakness and atrophy progressively worsened and spread to his whole body. He could not walk and became wheelchair-dependent five years after onset. He presented with severe scoliosis and pes cavus at the age of 13 years [Figures 1H and 1I]. When he was 15 years old, he became bedridden and was treated with non-invasive ventilation due to resting dyspnea. He died of cardiac arrest with a disease duration of 12 years and 4 months.
Patient 2 carried the reported de novo mutation within FUS (c.1574C>T, p.P525L) and was an 18-year-old girl who initially exhibited left arm weakness. Three months after onset, she had dysphonia and progressive weight loss. Four months after onset, she had difficulties in swallowing and drinking. Her sensation and cognition were not impaired. Neurological examinations revealed extensive muscle atrophy across her body. The thenar muscle, forearm muscle group, and gastrocnemius muscle on the left side showed moderate to severe atrophy, while those on the right side showed mild atrophy. The strength was 4/5 in the right limbs, 2/5 in the left upper limb, and 3/5 in the left lower limb. The deep tendon reflexes were depressed in the left limbs. EMG revealed a reduced CMAP of the right median (2.1 mV) and slightly reduced motor nerve conduction velocity. SNAP and sensory nerve conduction velocity were normal [Supplementary Table 2, https://links.lww.com/CM9/B339]. Fibrillation potential and sharp waves and prolonged polyphasic motor unit potentials were observed in the bulbar region, and acute denervation was observed in the cervical and lumbar regions. Five months after onset, she received gastrostomy and non-invasive ventilation. The patient died 13 months after onset.
Comparison of clinical features between JALS with SPTLC1 and FUS mutations
In total, 58 patients from 29 out of 32 articles met our inclusion criteria, including 16 JALS patients with SPTLC1 mutations [Table 1] and 42 with FUS mutations [Supplementary Table 3, https://links.lww.com/CM9/B339]. Most FUS mutations tended to cluster in exons 14 and 15 of FUS, while SPTLC1 mutations tended to cluster in exon 2 of SPTLC1. De novo mutagenesis is common in both genes. Clinical features such as AAO, onset site, respiratory failure, cognitive impairment, and disease duration were extracted manually for further analysis. The patients with SPTLC1 mutations tended to have an earlier AAO and the initial symptoms of lower limb spasticity and toe walking with longer disease duration (512.0 [416.7–607.3] months). Instead, most JALS patients with FUS mutations first presented with limb onset and had a rapidly deteriorating course (33.4 [21.6–45.1] months) [Table 2]. Both groups of patients suffered respiratory failure at the end of disease duration. Patients with FUS mutations also had a large proportion of bulbar onset (37.2%, 16/43), while those with SPTLC1 mutations only presented spinal onset. Sensory symptoms were presented in two JALS with SPTLC1 mutations (11.8%, 2/17) manifested as numbness and glove-stocking pain loss. However, sensory symptoms were not presented in JALS patients with FUS mutations, but other features, such as mental retardation and developmental delay, appeared more commonly. We found that the majority of JALS patients with SPTLC1 (88.2%, 15/17) and FUS (86.7%, 26/30) mutations presented with denervation on EMG. A small portion of JALS with FUS mutations (10.0%, 3/30) had prolonged central conducting times in motor-evoked potentials. Sensory involvement in EMG was presented in two patients with SPTLC1 mutations (11.8%, 2/17), consistent with their sensory symptoms. Three cranial magnetic resonance imaging abnormalities were presented in patients with FUS mutations (15.0%, 3/20), while there were none presented in those with SPTLC1 mutations.
Table 1 -
Clinical features of 17 JALS patients with
SPTLC1 mutations (including one patient reported in this study and 16 patients collected from 29 articles met our inclusion criteria).
| Patient |
Country |
SPTLC1 mutation |
Exon |
Inheritance pattern |
Gender |
AAO (years) |
Onset site |
Onset symptom |
Respiratory failure |
Sensory involvement |
Cognitive impairment |
Disease duration (years) |
EMG (Motor) |
EMG (Sensory) |
Cranial and spinal MRI |
Reference |
| 1 |
China |
p.A20T |
2 |
de novo
|
M |
7 |
Spinal |
Toe walking |
+ |
− |
− |
12.3 |
Axonal loss, denervation |
Normal |
NA |
Our study |
| 2 |
US |
p.A20S |
2 |
de novo
|
F |
5 |
Spinal |
LL spasticity |
+ |
− |
+ |
>15 |
Axonal loss, denervation |
Normal |
NA |
Johnson et al
[8]
|
| 3 |
US |
p.A20S |
2 |
de novo
|
F |
<10 |
Spinal |
LL weakness |
NA |
− |
+ |
∼10 |
Axonal loss, denervation |
Normal |
Normal |
Johnson et al
[8]
|
| 4 |
US |
p.S331Y |
11 |
de novo
|
F |
4 |
Spinal |
Toe walking |
+ |
+ |
− |
>7 |
Axonal loss, denervation |
Axonal loss |
Normal |
Johnson et al
[8]
|
| 5 |
Turkey |
p.L39del |
2 |
NA |
F |
15 |
Spinal |
UL + LL weakness |
− |
− |
NA |
>19 |
Denervation |
Normal |
NA |
Johnson et al
[8]
|
| 6 |
US and CA |
p.Y23F |
2 |
de novo
|
F |
4 |
Spinal |
LL spasticity |
+ |
− |
− |
>12 |
Axonal loss, denervation |
Normal |
NA |
Mohassel et al
[7]
|
| 7 |
US and CA |
p.Y23F |
2 |
de novo
|
F |
3 |
Spinal |
LL spasticity |
− |
− |
− |
>5 |
Axonal loss |
Normal |
NA |
Mohassel et al
[7]
|
| 8 |
US and CA |
p.F40_S41del |
2 |
de novo
|
F |
4 |
Spinal |
LL spasticity |
+ |
− |
− |
>24 |
Axonal loss, denervation |
Normal |
NA |
Mohassel et al
[7]
|
| 9 |
US and CA |
p.L39del |
2 |
de novo
|
M |
8 |
Spinal |
Toe walking |
+ |
− |
− |
>20 |
Axonal loss, denervation |
Normal |
NA |
Mohassel et al
[7]
|
| 10 |
US and CA |
p.L39del |
2 |
de novo
|
M |
3 |
Spinal |
Falls |
− |
− |
− |
>10 |
Axonal loss, denervation |
Normal |
NA |
Mohassel et al
[7]
|
| 11 |
US and CA |
p.A20S |
2 |
de novo
|
M |
3 |
Spinal |
Not able to run |
+ |
− |
− |
>18 |
Axonal loss |
Normal |
NA |
Mohassel et al
[7]
|
| 12 |
US and CA |
p.L39del |
2 |
AD |
M |
15 |
Spinal |
Ankle sprains |
− |
+ |
− |
>47 |
Axonal loss, denervation |
Axonal loss |
NA |
Mohassel et al
[7]
|
| 13 |
US and CA |
p.L39del |
2 |
AD |
F |
16 |
Spinal |
UL discoordination |
− |
− |
− |
>9 |
Axonal loss, denervation |
Normal |
NA |
Mohassel et al
[7]
|
| 14 |
US and CA |
p.L39del |
2 |
AD |
F |
14 |
Spinal |
Abnormal gait |
− |
− |
− |
>11 |
Axonal loss, denervation |
Normal |
NA |
Mohassel et al
[7]
|
| 15 |
US and CA |
p.L39del |
2 |
AD |
M |
10 |
Spinal |
Abnormal gait |
− |
− |
− |
>11 |
Axonal loss, denervation |
Normal |
NA |
Mohassel et al
[7]
|
| 16 |
US and CA |
p.L39del |
2 |
AD |
F |
6 |
Spinal |
Abnormal gait |
− |
− |
− |
>13 |
Axonal loss, denervation |
Normal |
NA |
Mohassel et al
[7]
|
| 17 |
China |
p.L38R |
2 |
de novo
|
F |
<10 |
Spinal |
LL weakness and falls |
+ |
− |
NA |
>0.2 |
Axonal loss, denervation |
Normal |
NA |
Lone et al
[9]
|
−: Absent; +: Present; AAO: Age at onset; AD: Autosomal dominant inheritance; CA: Canada; EMG: Electromyography; F: Female; JALS: Juvenile amyotrophic lateral sclerosis; LL: Lower limb; M: Male; MRI: Magnetic resonance imaging; NA: Not available; UL: Upper limb; USA: United States.
Table 2 -
Clinical features differ between JALS patients with
SPTLC1 and
FUS mutations (including two patients reported in this study and 58 patients collected from 29 articles met our inclusion criteria).
| Variable |
JALS patients with SPTLC1 mutations (n
= 17) |
JALS patients with FUS mutations (n
= 43) |
P-values |
| Gender ratio (male/female) |
0.6 (6/11) |
0.8 (19/24) |
0.53 |
| AAO (years, mean ± SD) |
7.9 ± 4.6 |
18.1 ± 3.9 |
<0.01 |
| Onset site, n (%) |
|
|
0.01 |
| Bulbar |
0∗
|
10 (23.3)†
|
|
| Bulbar and spinal |
0∗
|
6 (13.9)∗
|
|
| Spinal |
17 (100.0)∗
|
27 (62.8)†
|
|
| Disease duration (months, mean [95% CI]) |
512.0 (416.7–607.3) |
33.4 (21.6–45.1) |
<0.01 |
∗,†A same symbol indicates no statistical difference between two groups, and a different symbol indicates statistical difference between two groups. AAO: Age at onset; CI: Confidence interval; JALS: Juvenile amyotrophic lateral sclerosis; SD: Standard deviation.
To date, the majority of JALS patients with SPTLC1 mutations was Caucasian, and some of them have a positive family history; by contrast, most JALS patients with FUS mutations are Asian and have a negative family history. Therefore, to exclude the influence of different genetic backgrounds, we compared the clinical features among different races of JALS patients with SPTLC1 and FUS mutations and clinical features between JALS patients with de novo SPTLC1 and FUS mutations. The results were similar to the above results [Supplementary Tables 4 and 5, https://links.lww.com/CM9/B339].
Discussion
Juvenile patients with ALS have high phenotypic heterogeneity, and more than ten causative genes have been identified in JALS to date. FUS mutations are most frequently seen in JALS patients.[10] We identified seven unrelated JALS patients (43.8%, 7/16) carrying FUS mutations in our previous studies,[11,12] and the current study. Combined with the results of the literature review, there were 43 JALS patients carrying 17 different mutations in total. The majority of FUS mutations occurs in the C-terminus (exons 14 and 15), affecting the nuclear localization signal of FUS. Among them, mutation c.1574C>T (p.P525L) accounted for 44.2% (19/43). Previous studies have shown phenotypic heterogeneity in patients carrying different mutations within FUS.[11,14] However, Chen[15] demonstrated that JALS patients with p.P525L/p.Y526C mutations had no difference in AAO and disease duration compared to those with other FUS mutations.
In a German cohort, FUS mutations were detected in four JALS patients (66.7%, 4/6), and three of them had the same mutation of c.1574C>T (p.P525L) (75.0%, 3/4).[10] Similar results were verified in another Chinese JALS cohort. Zou et al[16] identified four FUS mutations in 16 JALS patients, and the mutation of c.1574C>T (p.P525L) accounted for half (50.0%, 2/4). In our cohort, FUS mutations were the most common genetic cause and among them, the mutation of c.1574C>T (p.P525L) accounted for 42.9% (3/7), which was consistent with reports worldwide. Other disease-causing genes have rarely been reported in JALS patients; therefore, more studies are needed to depict the genetic and mutation spectrum of JALS patients.
Recently, SPTLC1 was reported to be associated with JALS[7] which was first identified as a causative gene of HSAN1.[5] HSAN1-associated mutations were distributed in exons 5, 6, and 11, while ALS-associated mutations were mainly located in exon 2. Mutations in exon 2 enhanced SPT activity by weakening feedback inhibition of ORMDL3,[7,17] leading to disruption of sphingolipid homeostasis. JALS patients with SPTLC1 mutations present with lower limb spasticity manifesting as toe walking or gait abnormalities at an early age, commonly before the age of 10 years, which can be easily misdiagnosed as hereditary spastic paraplegia (HSP) at an early stage. However, diffuse lower motor neuron involvement and EMG showing extensive neurogenic damage help to differentiate between ALS and HSP. In this study, we identified a novel mutation within SPTLC1 (c.58G>A, p.A20T) in a sporadic patient with JALS. The patient with the SPTLC1 mutation exhibited lower limb spasticity and weakness without sensory or cognitive impairment at the age of 7 years, and symptoms spread progressively within a 12.3-year disease course. In addition, our patient presented symmetrically remarked muscle weakness in distal areas of the lower limbs accompanied by severe scoliosis and pes cavus, which might be caused by an onset of the distal lower extremity and long disease duration. The mutation (c.58G>A, p.A20T) in SPTLC1 was in exon 2 and identified as a de novo mutation. Altogether, the clinical or genetic characteristics of the patient were consistent with previous reports.[7,8]
After comparing the clinical features, we found that JALS patients with SPTLC1 mutations showed earlier AAO and longer survival than those with FUS mutations. Interestingly, patients with SPTLC1 mutations had no onset of bulbar, which may explain the mild progression they had, as patients with bulbar onset showed rapid progression.[18] These results help clinicians to distinguish the genotypes of JALS patients from their phenotypes at a clinical level, and genetic tests can support the diagnosis, which provides valuable prognostic information and therapeutic targets.
There are some limitations in this study. First, the sample size of newly recruited JALS was small. Furthermore, patients with SPTLC1 mutations were relatively fewer than those with FUS mutations. Finally, to comprehensively summarize the clinical features, five JALS cases with FUS mutations from three articles without a detailed phenotypic description (disease duration) were excluded during the literature retrieval. These limitations may affect the statistical results, which need to be confirmed by more studies.
In conclusion, we reported a novel SPTLC1 mutation in a Chinese JALS patient, which expands the mutational and phenotypic spectrum of JALS. In addition, we summarized that JALS patients carrying SPTLC1 mutations had an earlier AAO, longer survival and no onset of bulbar than those with FUS mutations, which helps to better understand the genotype–phenotype correlation of JALS.[19-40]
Acknowledgements
The authors sincerely appreciate the participants for their willingness to participate in this study.
Funding
This work was supported by the National Natural Science Foundation (No. 81671245).
Conflicts of interest
None.
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