Whole exome sequencing screen of ALS patients and pathogenicity classification of novel SOD1 mutations
From 2017 to 2021, a total of 8 different variants in SOD1 were found, including 6 known pathogenic variants (p.G38R, p.V48A, p.N87S, p.C112Y, p.I114T, and p.L145S) and 2 novel variants (p.G17H and p.E134*). Two heterozygous variants, c.49_50del insCA (p.G17H) and c.400G > T (p.E134*), were detected in two families with a positive family history (Fig. 1A and 1B), which was confirmed with sanger sequencing (Fig. 1C and 1D). Both novel variants were absent in ESP6500 and ExAC database, and were highly conserved from chimpanzee to zebrafish (Fig. 1E and 1F). The nonsense variant, p.E134*, generates the truncated protein caused by premature termination. However, the nonsense variant was not regarded as very strong evidence of pathogenicity because loss of function is not the primary mechanism in SOD1-related ALS. In addition, p.E134* was predicted to be detrimental by Mutation Taster and CADD. According to ACMG guidelines, the p.G17H variant was classified as likely pathogenic, whereas p.E134* was classified as variant uncertain significance (VUS).
To further elucidate the biological effects of these two variants, SOD1 protein and mRNA levels of mutants were markedly reduced compared to WT (Fig. 1G and 1H), indicating that the reduced protein expression was caused by reduced mRNA synthesis. The in vitro results showed that the mRNA levels of mutant SOD1 were degraded more rapidly than WT, suggesting a loss-of-function effect. The aggregation propensity assay showed that the cells overexpressing WT plasmid showed diffuse cytoplasmic SOD1 protein, while the misfolded aggregates were seen in cells transfected with p.G17H and p.E134* mutants (Fig. 1I). The soluble physiological SOD1 protein had a strong tendency to become toxic aggregates due to mutation, implying a gain-of-function effect. Therefore, the pathogenicity of the variant p.E134* was finally confirmed as likely pathogenic.
Mutation frequency of SOD1 in our ALS patients from Southeastern China
In this study, a total of 114 patients with SALS and 15 patients with FALS were screened by WES. Among them, 12 subjects were genetically identified as having SOD1 variants, 10 (10/15, 66.7%) of whom were FALS patients and 2 (2/114, 1.8%) of whom were apparently SALS patients. We previously reported 20 probands with SOD1 mutations in 36 unrelated FALS patients(6–9). When integrating these results into the current study, SOD1 mutations account for 58.9% (32/51) of our FALS cases.
A total of 21 SOD1 mutations, including 1 nonsense and 1 deletion/insertion mutations, were found spanning all five exons, but only one mutation was in exon 3 (Table 1). The most frequent SOD1 mutation was p.V48A (4/32, 12.5%) in 4 FALS probands, followed by p.H47R, p.C112Y, and p.G148D (each were found in 3 probands (3/32, 9.4%)), as well as p.H121Q, p.L145S (each were found in 2 probands (2/32, 6.3%)), and the remaining mutations were each found in 1 proband (Fig. 1J). Among all the mutations, p.L145S, p.P75S, and p.I114T are found in sporadic ALS patients (Fig. 1J).
Table 1
The clinical features of the probands carrying mutations in SOD1
Probands | Variant | Exon | Gender | Family History | AAO (y) | Diagnostic Delay (m) | Site of Onset | Side of Onset | Disease Duration (m) | Predominant Features |
1 | A5S | 1 | M | F | 29 | 3 | LL | L | 12 | LMN dominance | |
2 | A5V | 1 | F | F | 47 | 8 | UL | L | 15 | Classical ALS | |
3 | G11V | 1 | F | F | 25 | 9 | LL | L | 15 | Classical ALS | |
4 | G17C | 1 | M | F | 49 | 8 | spinal | L | 38 | LMN dominance | |
5 | G17H | 1 | M | F | 23 | . | LL | R | 36 | Classical ALS | |
6 | F21C | 1 | M | F | . | . | . | . | . | . | |
7 | G38R | 2 | M | F | 40 | 12 | spinal | Both | > 156 A | Classical ALS | |
8 | H47R | 2 | F | F | 58 | 84 | UL | R | 180 | LMN dominance | |
9 | H47R | 2 | M | F | 53 | 18 | LL | Both | 120 | LMN dominance | |
10 | H47R | 2 | F | F | 55 | 18 | LL | Both | > 144 | LMN dominance | |
11 | V48A | 2 | F | F | 53 | 20 | LL | R | 17 | LMN dominance | |
12 | V48A | 2 | F | F | 42 | 12 | UL | Both | > 30 A | LMN dominance | |
13 | V48A | 2 | F | F | 46 | 13 | LL | R | > 27 A | Classical ALS | |
14 | V48A | 2 | M | F | 64 | 12 | LL | Both | > 25 A | LMN dominance | |
15 | P75S | 3 | M | S | 59 | 23 | LL | Both | 65 | LMN dominance | |
16 | D84G | 4 | M | F | 32 | . | LL | R | 70 | LMN dominance | |
17 | N87S | 4 | M | F | 72 | 11 | UL | R | > 21 A | Classical ALS | |
18 | L107V | 4 | M | F | 41 | 2 | LL | R | 10 | Classical ALS | |
19 | C112Y | 4 | M | F | 47 | 9 | UL | R | 53 | LMN dominance | |
20 | C112Y | 4 | M | F | 50 | 15 | LL | R | 40 | LMN dominance | |
21 | C112Y | 4 | M | F | 47 | 6 | LL | R | 60 | LMN dominance | |
22 | I114T | 4 | M | S | 60 | 10 | UL | R | 21 | Classical ALS | |
23 | H121Q | 5 | F | F | 42 | 11 | LL | Both | 51 | LMN dominance | |
24 | H121Q | 5 | F | F | 60 | 7 | UL | L | 39 | Classical ALS | |
25 | E134* | 5 | M | F | 48 | 6 | UL | R | > 34 A | Classical ALS | |
26 | L145S | 5 | F | F | 50 | 6 | LL | Both | > 31 A | LMN dominance | |
27 | L145S | 5 | F | S | 48 | 28 | LL | L | > 46 A | Classical ALS | |
28 | C147R | 5 | F | F | 39 | 3 | bulbar | . | 9 | Classical ALS | |
29 | G148D | 5 | F | F | 34 | 4 | UL | Both | 12 | LMN dominance | |
30 | G148D | 5 | F | F | 37 | 6 | UL | L | 15 | Classical ALS | |
31 | G148D | 5 | F | F | 38 | 12 | LL | L | 14 | Classical ALS | |
32 | I150T | 5 | F | F | 37 | 7 | LL | Both | 12 | Classical ALS | |
Abbreviation: AAO = age at onset; UL = upper limb; LL = lower limb; LMN = lower motor neuron |
Clinical features and natural history of ALS patients carrying the SOD1 mutation
Combining our previously reported SOD1 mutated patients in our centers, a total of 32 probands were genetically identified with SOD1 mutations (Table 1). 29 (29/32, 90.6%) of the probands were FALS patients, and 3 (3/32, 9.4%) were apparently SALS patients. The gender ratio (M: F) was 1:1. With the exception of 1 case with missing data, only 1 patient among 31 available probands exhibited bulbar onset. The majority of them (18/31, 58%) presented with lower limb onset, but some (10/31, 33.3%) presented with upper limb onset. Sixteen probands presented with a predominantly lower motor neuron phenotype, while another 16 probands were the classical ALS phenotype.
With the exception of the patient with missing data, 31 patients had available AAO data for analysis. The mean (SD) AAO was 46 ± 11.4 years, and the median AAO was 47 years (Fig. 2A). The mean AAO of patients carrying mutations in exon 1 [n = 5, 34.6 (12.4)] was statistically significantly earlier than those with mutations in exon 2 [n = 8, 51.4(8.2)] (p = 0.038) (Fig. 2B). There was no difference in the mean (SD) AAO between male and female patients [47.6 (12.2) vs 45.25 (11.2), p = 0.58] (Fig. 2C). The onset of FALS in patients is earlier than that of SALS, albeit without statistical significance [45.4 (11.4) vs 55.7 (10.2), p = 0.15] (Fig. 2D). The AAOs of ALS patients were highly heterogenous with the 25th and 75th percentiles at 38 and 53 years, respectively. Six mutations (p.A5S, p.G11V, p.G17H, p.D84G, p.G148D, p.I150T) showed relatively younger AAOs, which was lower than the first quartile (25%, ༜38 years) and 6 mutations (p.H47R, p.V48A, p.P75S, p.N87S, p.I114T, p.H121Q) presented with relatively older AAOs, which were higher than the third quartile (75%, > 53 years). The youngest and oldest AAOs are p.G17H and p.N87S, which were found in patients in their twenties and seventies, respectively.
Twenty-nine probands patients were accessible for data regarding diagnostic delay. With the exception of one case with an extremely long diagnostic delay of 84 months, the mean (SD) diagnostic delay of the remaining was 10.7 ± 6.2 months (Fig. 3A). The diagnostic delay showed no differences in different exons (Fig. 3B). The time to diagnosis of male patients exhibited no difference compared to female patients [10.4 (5.9) months vs 11.0 (6.7) months, p = 0.82] (Fig. 3C). It is worth noting that the mean of the diagnostic delay of FALS patients is less than that of SALS patients, and was found to be statistically significant [9.5 (4.8) months vs 20.3 (9.3) months, p = 0.0026] (Fig. 3D). Diagnostic delay varied greatly across different mutations. Four mutations of 4 probands, namely p.A5S, p.L107V, p.C147R, and p.G148D, showed a diagnostic delay less than the first quartile (25%, ༜6 months). Another 5 mutations of 8 probands, namely p.H47R, p.V48A, p.P75S, p.C112Y, and p.L145S showed a diagnostic delay more than the third quartile (75%, > 12 months).
Disease duration data were available for 31 probands, and 9 patients still survive at the censoring date. The median survival time was 40.0 months and the 5-year survival rate was 30.7% for all subjects (Fig. 4A). The survival time showed no difference between all male and female patients (Fig. 4B). Given the extremely long survival time in mutation of p.H47R, we excluded the p.H47R mutation in both male and female patients for statistical analysis. Strikingly, male patients showed significantly longer survival time than female patients (40 months vs 16 months, p = 0.05) (Fig. 4C).