Genetic Landscape of Amyotrophic Lateral Sclerosis in Czech Patients

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a heterogeneous group of neurodegenerative conditions, comprising several clinical variants, all leading to various degrees of progressive upper and lower motor neuron loss. Moreover, other related neuronal populations can be affected simultaneously (frontotemporal circuits leading to cognitive disorder or frontotemporal dementia, extrapyramidal system etc.) so that ALS can be regarded as a multisystem degeneration [1]. The pathogenesis of ALS is not fully understood, but there is a genetic component. In 5–10% of patients, there is a family history of ALS (or frontotemporal dementia), usually with autosomal dominant (AD) inheritance [2]. Currently, more than 30 genes have been linked to the pathogenesis of ALS [3]. Mendelian gene variations have been identified in about 50% of those with a family history (fALS) [4, 5] and about 11% of those without, classified as sporadic ALS (sALS) [6]. Every familial ALS gene variant has also been seen in patients with no obvious family history. Furthermore, fALS is often clinically indistinguishable from sALS and even shows the same neuropathological pattern [2]. The most common genetic cause of ALS is an expansion of a GGGGCC (G4C2) – hexanucleotide repeat (HRE) in the first intron of the C9orf72 gene, with a frequency of 33.7% in fALS and 5.6% in sALS among the European population [4], but with some variation among ethnic groups of different ancestry [7–10]. The next most common causal mutations have been found in SOD1, TARDBP, and FUS genes, both in patients with fALS and those with sALS [4]. Furthermore, more than one pathogenic or likely pathogenic variant carried simultaneously by a single individual, suggesting possible oligogenic interaction, has been documented in up to 3% of patients in a recent large-scale study [6].

While robust genetic data regarding mutation frequencies in fALS [4, 5] as well as sALS [6, 7] patients in Europe exist, ethnics with genetic ancestry different from that in the Czech population have been assessed (e.g. German, Dutch, Italian, Portuguese etc.). So far, only one study from a population with predominantly Slavic ethnicity has been published [10], where only four major ALS-associated genes have been assessed. It has been shown that individuals from different populations carry different profiles of rare and common variants and that low-frequency variants show substantial geographic differentiation [11]. Notably, the frequency of HRE in C9orf72 in sALS varies from 0% to 21% [12] and could be specific in the Czech population. Our work could fill a gap in the genetic diversity of the European ALS population.

Thus, considering the abovementioned situation, we designed a study analyzing the genetic architecture in Czech familial, as well as sporadic ALS patients, assessing the frequency of HRE in C9orf72 and also of rare variants in 36 ALS-linked genes via next-generation sequencing in our cohort. With the advance of gene-specific therapies (antisense oligonucleotides; ASOs) specifically for SOD1 and C9orf72-linked ALS, these data will have an increasingly important clinical impact.

MATERIALS AND METHODS

Patients

This was a two-centre, two-cohort study. We recruited 88 Czech patients diagnosed with ALS (9 familial and 79 sporadic cases) according to the revision of the El Escorial criteria – 2015 [13], 71 of them at the European Reference Network (ERN) Neuromuscular Center (NMC), University Hospital Motol and 17 of them at the ERN NMC, University Hospital Brno. The diagnosis of ALS was ascertained by an experienced neuromuscular neurologist (DB, RM, EV) after alternative diagnoses had been excluded. Diagnosis of comorbid frontotemporal dementia was ascertained according to Strong criteria [14]. Familial ALS cases were defined as patients with at least one first or second-degree relative with ALS or FTD. We recorded relevant clinical and demographic features for all patients (Table 1). The study design comprised two distinct cohorts. First one was a prospective longitudinal cohort where we recruited patients and

Table 1

Patient demographics and clinical characteristics (n = 88)

Age (years)	51.2 (19; 82)
Young-onset patients	24 (27.3%)
Sex
Male	50 (56.8%)
fALS	9 (10.1%)
Site of onset
Spinal	68 (77.3%)
Subtypes
Classic ALS	57 (83.8%)
LMN-predominant	9 (13.2%)
With FTD	2 (3.0%)
Bulbar	20 (22.7%)
Subtypes
Bulbar only	15 (75.0%)
with FTD	5 (25.0%)

ALS – amyotrophic lateral sclerosis, Classic ALS – upper and lower motor neuron involvement, fALS – at least one first- or second-degree relative with ALS and/or FTD, FTD – frontotemporal dementia, LMN – lower motor neuron; young-onset:<45 years; Continuous data are presented as median (min; max), categorical data as absolute numbers and relative frequencies.

analyzed their samples from 2015 to 2020 in a research setting (n = 52, all at the Neuromuscular Center, Department of Neurology and Department of Biology and Medical Genetics, University Hospital Motol). The second cohort was a retrospective one, enriching the dataset with samples from patients referred for routine genetic testing, mainly due to family history, young age of onset (<45 years) [15] or associated dementia (ERN NMC University Hospital Motol n = 19, ERN NMC University Hospital Brno n = 17; all 36 samples analyzed at the Centre of Molecular Biology and Genetics, University Hospital Brno) (Fig. 1).

Fig. 1

Schematic of patient distribution and variant detection between and within two cohorts. Prospective cohort – longitudinal recruitment and sample analysis between 2015–2020 at the ERN-NMC and Department of Biology and Medical Genetics, both University Hospital Motol; Retrospective cohort – samples from patients, analyzed in a routine setting, all at the Centre of Molecular Biology and Genetics, University Hospital Brno; 1 – pathogenic mutations in other ALS-linked genes: FUS, TBK1; 2 –TARDBP; fALS – at least one first- or second degree relative with ALS and/-or FTD.

Whole blood for DNA extraction was obtained following informed written consent. We obtained approval for this study from the Institutional Ethics Committee of University Hospital Motol.

RESULTS

For the sake of data presentation clarity, we combined the results from the two cohorts together since the applied criteria for variant classification were identical.

DISCUSSION

To the best of our knowledge, this was the first comprehensive assessment of the genetic landscape of ALS in Czech patients. We aimed to bring pilot data on the genetic findings among familial and sporadic ALS patients from our country. Familial ALS cases were slightly overrepresented in our study (10.1%) due to the intentional enrichment of these individuals in the retrospective cohort. As this was not a population-based study, this percentage probably does not reflect the actual proportion of fALS cases in the Czech population (based on our estimates, the actual proportion will be around 5% of the ALS population). The unusually high proportion of pathogenic mutations detected in our fALS cases (88%) stems probably from the small sample size and would rather be lower in larger cohorts of patients, as other studies documented [4, 5]. On the other hand, the proportion of cases with pathogenic mutations among sALS (10.1%) was very similar to a recent, large-scale study (6013 sALS cases) [6] and could, therefore, be a realistic representation of the situation in the Czech population.

The most frequent mutation was the C9orf72 repeat expansion, detected in 12 patients (13.5%) from the entire study population. This data is in line with many previous cohorts. Six out of the 12 patients with the C9orf72 HRE had no family history of ALS or FTD, making this mutation the most common finding among sporadic cases (7.6%). Again, the proportion was relatively similar to other cohorts from European populations [6, 7]. With regards to the clinical phenotype of the C9orf72 HRE carriers, bulbar onset significantly predominated (seven out of 12 patients, 58.3%) – which is in line with findings from substantially larger cohorts from Europe [27, 28] – and frontotemporal dementia cases were overrepresented (3 out of 12 patients, 25%), compared to the rest of the study population, although the latter difference was not statistically significant. Notably, among sALS patients, the presence of ALS-FTD phenotype was associated with a significantly higher likelihood of detecting a pathogenic variant. However, the generalizability of this finding is limited due to the small size of our study population.

Young-onset ALS patients were overrepresented in our study (27.3% of the entire cohort, compared to about 10% referred in the literature [15]). Contrary to expectations, based on predictions from a validated model of pathogenesis [29], as well as findings from larger cohorts [30], the detection rate of pathogenic (or likely pathogenic) variants in this subgroup was not higher than in the rest of the study population. Notably, there were four young-onset patients in our study, carrying five variants of uncertain significance in FIG4 p. (Arg699Cys), SETX p. (Phe1599Ser), SOD1 p.(Asp126Glu), TARDBP p.(Val57Ile) and TBK1 p.(Met690Thr), where pathogenicity seems possible, but could not be confirmed. One plausible explanation for the low detection rate within the young-onset group could have been the overall small population of our study. Nevertheless, even with the overrepresentation of young-onset patients, most of the pathogenic variants were detected in the group with standard age of onset, suggesting a possibility of a missing genetic variability in the former subgroup. Interestingly, another study focused on young-onset sporadic patients reported a low detection rate of pathogenic mutations in established ALS-linked genes in this subgroup as well [31]. The authors focused on the burden of rare variants in genes coding RNA-binding proteins that are not yet established as causal in ALS pathogenesis but are involved in common functional pathways. This study gave some evidence for oligogenic interaction and synergistic effect among variants in this functional cluster of genes in the group of young-onset patients. As such a broad gene analysis was beyond the scope of our study, which was limited mainly by the sample size, we can only speculate about the applicability of this explanation to our cohort. Nevertheless, this seems to be a promising direction to elucidate the so far unexplained genetic component among young-onset patients in our cohort.

Mutations in the FUS and TARDBP genes are among the more commonly detected in ALS patients [4, 6]. Besides the two documented pathogenic variants, detected in two fALS patients from our cohort – p.(Arg521Cys) in the FUS gene and p.(Ala382Thr) in TARDBP - we found a novel variant of uncertain significance in TARDBP, p.(Val57Ile). This variant was detected in a sporadic young patient with a LMN-predominant phenotype and was inherited from her unaffected mother. Interestingly, this affected individual harboured a second heterozygous variant, a likely pathogenic, autosomal recessive missense mutation p.(Asp126Glu) in SOD1, inherited from her unaffected father. Although the segregation analysis ruled out a causal effect of neither of these variants, we speculated a possible synergistic effect, when present simultaneously in one individual, based on documented interaction between SOD1 and TDP-43 proteins in an experimental study [32]. Notably, an oligogenic interaction with other co-segregating rare variants has been documented for another variant in SOD1, p.(Asp91Ala) [33], which is also remarkable for its reduced penetrance and variable inheritance (recessive or dominant) [34].

We also noticed the absence of causal mutations in SOD1 among fALS, as well as sALS patients in our cohort, which very probably stems from its small sample size., Data from a large-scale study documented SOD1 mutation detection in only 0.63 % of sporadic ALS patients [6]. The size of our fALS cohort was small and unequally represented geographic regions of our country. Thus, a regional family cluster of SOD1-positive cases could have easily been missed.

In the last decade, several studies brought evidence for a suggested oligogenic model of inheritance in ALS [35, 36]. On the other hand, some studies documented the C9orf72 HRE alone to be sufficient to cause ALS, irrespective of the co-occurrence of other variants in ALS-linked genes [37, 38]. In our study, besides the abovementioned young-onset patient harbouring variants both in SOD1 and TARDBP, we further detected a variant p.(Ser262*) in OPTN in two related patients (mother and daughter), both harbouring C9orf72 HRE. This variant in OPTN has been detected in an autosomal recessive form of ALS only [23]. As a matter of fact, the phenotype differed to some degree between the two patients (much younger age of onset in the daughter, the presence of FTD in the mother, but not in the daughter). However, since they were both harbouring the same OPTN variant, this seems not to be a plausible genetic modifier and we therefore deemed its causal significance in our patients as rather unlikely.

We identified a novel variant in the NEK1 gene p.(Glu254*) in a sporadic, bulbar onset patient. For this variant p.(Glu254*), we assume the loss of function (LoF) effect. The LoF variants in the NEK1 gene, coding NIMA-associated kinase, have been suggested to play a direct pathogenic role by some authors [39], while others consider them to only confer significant susceptibility to disease development and hypothesize a necessary oligogenic interaction with other co-segregating rare variants in ALS-linked genes [40, 41]. In our patient, we haven't found any such additional variant in the extent of examined genes. Taken together, we categorized this variant as of uncertain significance.

Furthermore, we detected two variants in TBK1 in two sporadic patients with spinal onset ALS without FTD. First, a previously recorded [20], likely pathogenic splice-donor variant c.540 + 1G > T, which probably disrupts the donor splice site and could lead to haploinsufficiency via loss-of-function mechanism – a likely molecular genetic mechanism of toxicity in TBK1 mutations. The other one was a novel, missense variant p.(Met690Thr), assessed by in silico analysis as of uncertain significance. While the mechanism through which nonsense mutations lead to pathogenesis via haploinsufficiency has been elucidated, the mechanisms underlying the disease due to missense mutations are still largely unclear [43]. Our data add to the growing knowledge that mutations in TBK1 are an important contributor to the genetics of sporadic ALS.

Moreover, we identified two variants in FIG4. Although biallelic pathogenic variants in FIG4 have been established as causative for AR-Charcot-Marie-Tooth disease 4J, several studies have brought some (case level) evidence of its involvement in the pathogenesis of autosomal dominant ALS type 11 [21, 35, 44]. The variant p.(Ile41Thr) in the heterozygous state has previously been reported in cohorts from European [21] and Northern American [30] populations and is reported in ClinVar with conflicting results. However, due to its relatively high prevalence in the gnomAD control database (0.002), we do not think that it alone causes ALS in accordance with the conclusion from another recent study [44]. The other detected variant in FIG4- p.(Arg699Cys) – has been reported in a German fALS cohort [5]. Without co-segregation data, it could not be conclusively confirmed as disease-causing.

Further, we found two novel missense variants in SETX, a gene associated with early-onset, autosomal dominant ALS type 4, in two sporadic patients. This rare form of ALS has been described as a very slowly progressive one without the involvement of respiratory or bulbar muscles [45]. Notably, a broader phenotypic variation among ALS patients with novel SETX variants has been reported in more recent studies [46]. The patient with the p.(Phe1599Ser) variant was a young-onset (38 years) sporadic case with a marked upper-motor neuron involvement, developing a prominent bulbar involvement later on in the course of the disease. The other patient, carrying the variant p.(Ser2289Pro) presented with sporadic ALS with onset at 60 years and a rapid course of the disease. Without segregation analysis and further evidence, we cannot conclude regarding the pathogenicity of these variants. Analogically, this was the case of a rare variant p.(Pro494Leu) in UBQLN2, detected in a sALS patient in our cohort and previously described in a French fALS patient [26].

We detected two previously recorded, likely pathogenic frameshift mutations in SPG11 in a heterozygous state: p. (Leu920Thrfs*10) and p.(Glu1026Argfs*4) [24, 25] and a novel missense variant p.(Ser3Phe) in SPAST. Although mutations in both SPG11 and SPAST have been associated with hereditary spastic paraplegia, biallelic variants in SPG11 are also causal in recessive juvenile ALS [47]. Furthermore, heterozygous variants in SPG11 have been reported from cohorts of fALS and sALS patients [48, 49]. Nevertheless, the latter studies haven't established any causal relationship between these novel variants and ALS. Association between SPAST and ALS has been reported only on a case level so far [50, 51]. As for the variants in SPG11, detected in our cohort, it is also possible that we failed to reveal the second variant (e.g. deep-intronic or CNV). However, the phenotype of the patients (onset > 60 years, bulbar / LMN-predominant sALS) does not resemble previously described cases. The clinical significance of these variants is, therefore, uncertain.

We are aware of the limitations of our study due to the different approaches to library preparation and, thus, different coverage of relevant ALS genes between the two laboratories. The small size of the patient cohort may have led to a possible omission of variants in less common ALS-associated genes and limits the generalizability of the phenotypic features, observed in our cohort, due to low statistical power. Moreover, as a result of the hospital-based design of our study, as well as of the intentional enrichment of specific subgroups of patients (young-onset, fALS), the obtained data might be skewed and not entirely representative regarding the frequency and proportion of the most common variants, as compared to a population-based study.

In summary, our data has shown the frequency of C9orf72 HRE among Czech ALS patients to be very similar to other European populations. Furthermore, the data suggest some consistent phenotypic features (bulbar onset or predominance, higher proportion of FTD) even among apparently sporadic patients with the C9orf72 HRE, providing altogether (regarding its relatively high prevalence) some guidance for targeted first-line genetic testing. Pathogenic mutations in TBK1, FUS, and TARDBP, commonly detected among other cohorts, are present in the Czech population as well. On the other hand, we detected no causal variants in SOD1 in our study. We encountered a low yield of pathogenic or likely pathogenic mutations among young-onset patients in our cohort, contrary to our assumptions, upon which we intentionally recruited this subgroup. From another perspective, these findings suggest a high probability of a missing genetic variability at the level of as yet not established (and, therefore, not examined in our study) genes. In our opinion, exploring this subgroup of patients for new groups of genes is highly warranted, holding a promise of a novel option for potential gene-modifying treatment in ALS. Regarding the variants of uncertain significance found in patients of our cohort, no definite conclusion regarding their pathogenicity can be drawn, and further functional, genetic, and clinical studies in large independent cohorts are required to fully understand and establish their contribution to ALS.