Role of genetics in amyotrophic lateral sclerosis: a large cohort study in Chinese mainland population

Background

Amyotrophic lateral sclerosis (ALS) is one of the most common incurable neurodegenerative diseases that primarily affect the motor neurons, eventually resulting in progressive paralysis and death 3–5 years after disease onset.1 Approximately 5%–10% of patients with ALS have a family history (FALS), while the remaining cases are sporadic (SALS).2

To date, variants in more than 120 genes have been reported to be implicated in ALS (https://alsod.ac.uk). Variants in ~40 genes have been shown to cause or significantly increase the risk of ALS, although the pathogenicity of variants in some of the genes remains to be validated. Among them, C9ORF72, SOD1, FUS and TARDBP are the most common causative genes, accounting for 54.5% of FALS and 7.5% of SALS in Caucasians.3 Genetic studies of ALS in other populations have been reported, but largely in a small number of ALS genes and in small-sized cohorts. Comprehensive genetic studies of large ALS cohorts with unbiased approaches, such as whole-exome sequencing (WES), are limited.

In the current study, using repeat-primed PCR (RP-PCR) and WES, we studied a total of 1587 patients with ALS and 1866 inhouse controls from a cohort of Chinese population, which represents one-fifth of the world’s population, to characterise the genetic and clinical spectrum of ALS in Chinese population and provide essential information for ALS research and clinical practice. The study has the following aims: (1) systematically identify pathogenic and likely pathogenic (P/LP) (for causative genes) or deleterious (for new, needing to be confirmed or risk genes) variants of 41 known ALS-associated genes in an unprecedented large cohort of Chinese patients with ALS, summarise and compare the mutation frequencies of FALS and SALS, and perform gene/allele-based burden analyses to reveal whether rare variants of these genes contribute collectively to ALS; and (2) investigate the genotype–phenotype correlation in Chinese patients with ALS, which will benefit genetic counselling, early diagnosis intervention and prognostic assessment.

Discussion

In the current study, we determined the genetic spectrum of 41 ALS-related genes and the genotype–phenotype correlation based on a large Chinese ALS cohort. Furthermore, for the first time, we systematically analysed the collective risk of rare P/LP or deleterious variants in these genes, which led to a better understanding of their pathogenicity in ALS.

To date, several genetic studies have comprehensively analysed the mutation spectrum of ALS using next generation sequencing (NGS) methods in European patients,5 18–21 providing important information for developing guidelines for genetic testing for ALS in clinical practice. In Asia, especially in China, which contributes a fifth of the world’s population, only two NGS studies on a limited sample size of patients with ALS16 22 have been performed to analyse the mutation spectrum of ALS-associated genes (45 and 268 patients with ALS involved, respectively). The small-sized studies prevented the clarification of genetic spectrum and the genotype–phenotype correlation. In addition, the clinical features of the consecutive patients involved in this study, including the proportion of FALS, sex ratio, initial symptoms, mean age of onset and median survival time, were consistent with other studies from Chinese population,22 23 although some of which were slightly different from patients from Caucasian populations (eg, the proportion of patients with FALS was 1.2%–2.7% in Chinese23 (4.0% in our cohort), but 5%–10% in Caucasians2; and the mean age of onset was 54.4 years old in a larger Chinese ALS cohort24 (53.9 years old in our cohort), but 62.1–66.3 years old in Caucasians25). Therefore, it was reasonable that the ALS cohort involved in this study could be a typical representative of the Chinese ALS population, and these differences of clinical features between Chinese and Caucasian patients indicated it is necessary to unearth the genetic features from other ethnicities.

In the present study, we comprehensively analysed the mutation spectrum of known causative and risk or needing to be confirmed ALS genes in Chinese patients with ALS, which were classified into two categories (GI and GII). We found the mutation frequencies of the 16 GI genes were as high as 40.6% and 8.6% in FALS and SALS, respectively, which were consistent with those reviewed in previous studies.23 26 According to our comprehensive analysis, we suggest a genetic screening strategy in Chinese patients with ALS: in FALS, the following genes should at least be analysed, SOD1 (21.9%), FUS (6.3%) TARDBP (3.1%) and VCP (3.1%), which account for 34.4% of patients, and in SALS, SOD1 (1.90%), C9orf72 (1.31%), NEK1 (1.12%), FUS (0.98%), TARDBP (0.85%) and TBK1 (0.66%), which account for about 6.8% of patients. Compared with mutation frequencies in patients with ALS of European ancestry,27 SOD1 is the most common causative gene in Chinese patients with FALS and SALS.26 Surprisingly, G4C2 repeat expansion in C9orf72 is the second common causative gene for SALS in our cohort, which was substantially higher than that in other Chinese cohorts (1.31% vs 0%–0.3%).28 29 However, no patients with C9orf72 G4C2 repeats had reported family history of ALS in this study, but up to 40% of C9orf72 positive FALS from Europe and the USA were identified,30 which might be partly accounted by deficient awareness of ALS in Chinese populations in the last few decades and the age-dependent penetrance of C9orf72 G4C2 repeats.30 The mutation carriers of C9orf72 G4C2 repeats from our cohort were suggested to share a common founder of European ancestry,31 indicating that it is an ideal candidate cohort for prospective studies investigating the role of C9orf72 in the Chinese population due to its age-dependent penetrance,30 and the upcoming targeted therapies for C9orf72 repeat will also benefit a subset of Chinese patients with ALS. In addition, more than 33% of patients whose age of onset was <30 years carried P/LP variants, whereas the mutation frequencies (about 11% or 8%) presented plummeting in patients whose age of onset was >30 years, which supported the hypothesis that the number of multistep process of ALS (usually six-step process)25 will be reduced in patients with ALS with genetic mutations compared with those without mutations.32 According to our findings, varied mutation frequency and spectrum among different groups of age of onset suggest that patients who are younger than 30 years should regularly undergo genetic testing to clarify the aetiology since high mutation frequencies especially for FUS were seen. Moreover, our finding also suggested that ageing and environmental factors play a more important role than genetic factors in elderly patients with ALS. Therefore, these findings might contribute to drafting a genetic scanning strategy for Chinese patients with ALS.

Our study further clarifies the correlation between genotypes and phenotypes of ALS in the Chinese population. Generally, significantly earlier age of onset, faster progression and shorter median survival time were found in patients carrying P/LP variants than in patients without P/LP variants, which was consistent with other studies and suggests that causative genetic variants are a poor predictor of outcome of ALS.1 31 However, we did not find much more proportion of patients beginning with bulbar onset in patients with the GI gene variants compared with patients without GI/GII variants because the former were generally considered a more ‘severe’ phenotype. Nearly half of patients with variants of the three genes (75 patients out of 155 patients with the GI gene variants), namely SOD1, TARDBP and NEK1, variants which will always be linked with spinal onset (as shown in our study; online supplemental table S6), might contribute to the current finding. Although phenotypic pleiotropy of ALS genes was suggested, genotype–phenotype correlation analyses further determined the main clinical manifestations of each gene. As such, patients with SOD1 variants tend to first involve lower limb. Mutations in FUS and TARDBP were associated with an earlier age of onset, faster progression and shorter survival, but a reverse relationship was found in patients with variants of NEK1 and ANXA11. Most patients with P/LP variants of C9orf72 likely presented with cognitive and frontal behaviour impairments, but relatively less in SOD1. Importantly, more clear and sharing phenotypes of each P/LP variant were characterised in our study, such as p.Leu39Val, p.His47Arg and p.Pro67Ala in SOD1, which were associated with slower progression, and p.Cys112Tyr in SOD1, p.Gly294Val in TARDBP and p.Arg521His in FUS, which were associated with faster progression. Because all of the patients came from different families and most shared variants were suggested to come from a founder effect based on our haplotype analysis, our results suggest that these causative genetic variants strongly modify disease phenotypes.

Our study further supports the findings that the most common variants in Caucasians, p.Ala4Val of SOD1 and p.Arg521Cys of FUS, are associated with faster progression,33 34 although the former was found in only one late-onset patient whose survival duration was about 14 months. However, the most common variants of TARDBP, p.Ala382Thr, reported in Caucasian patients with ALS,35 and p.Met337Val, reported in Chinese from Eastern China,16 were not found in our cohort. Another variant, p.Gly294Val, of TARDBP was first reported in Chinese and was the most common variant in our cohort. Its pathogenicity was previously suggested by experimental evidence from patient-derived induced pluripotent stem cells36 and is supported by our functional experiments (online supplemental figure S3). Our haplotype analysis was also consistent with a common ancestral origin reported in five Italian and Moroccan patients with ALS sharing a haplotype interval of 1.4 Mb.15 However, whether patients with this variant identified in our cohorts and Caucasians came from a founder effect was unknown due to differences in polymorphisms used for haplotype analysis. In any case, our results suggested it is necessary to explore the genetics of patients with ALS from different ethnicities, even from different regions in the same ethnicity, although some patients might share a few P/LP variants.

Since 2014, 12 new ALS causative/risk genes have been discovered (shown in online supplemental table S1), namely CHCHD10, TBK1, NEK1, ANXA11, KIF5A, DNAJC7, MATR3, TUBA4A, CCNF, TIA1, GLT8D1 and CYLD; only the former six genes were classified as ALS causative genes due to strong evidence (https://alsod.ac.uk/). The remaining 6 genes and other 19 ALS risk genes require replication or resolution of conflicting evidence or have not been well studied in Chinese population. However, we did not find significant enrichment of rare variants by burden analysis at the gene level, which did not support its pathogenicity in ALS. However, the results should be interpreted with caution. In our analysis, consensus definition for P/LP or damaging variants in patients with ALS and inhouse controls was performed; however, the pathogenicity predicted to be deleterious variants needs to be confirmed in future studies. In addition, aetiological heterogeneity, incomplete penetrance, late-onset diseases or limited sample size make it difficult to detect the associations between rare variants and the disease in case–control studies. As shown in the results, rare P/LP variants did not significantly enrich in each causative gene, except for SOD1, FUS, TARDBP, OPTN and UBQLN2.

The current study represents a comprehensive and systematic screening of ALS-associated genes in an unprecedented large cohort of patients with ALS from West China; however, it has some limitations. First, we focused on the mutation spectrum and conducted genotype–phenotype correlation analyses in the GI genes, but the pathogenicity of some GII genes in ALS still needs to be confirmed in future studies. Second, we only analysed the clinical and genetic architectures of patients with putative rare P/LP variants in the coding region of ALS-causative genes, but not of those with VUS. Although the pathogenicity of variants was determined according to the ACMG standard, their pathogenicity needs to be confirmed in more functional studies. Variants in non-coding regions, such as regulatory elements, which may play important roles in ALS, were not analysed in the current study.

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