SOD1 Variants in Patients With Amyotrophic Lateral Sclerosis in Central Eastern Europe: From Genetic Testing to SOD1 Targeted Therapy

ABSTRACT

Background

Amyotrophic lateral sclerosis (ALS) is one of the most devastating fatal motor neuron diseases, characterized by progressive degeneration of motor neurons in the brain and spinal cord. A significant advance in ALS therapy was achieved with the recent European Medicines Agency approval of Tofersen, the first antisense oligonucleotide (ASO) specifically targeting SOD1 mRNA, a key genetic determinant of the disease. Yet, despite its clinical relevance, data on SOD1‐ALS in Central Eastern Europe remain scarce.

Methods

Here, we present a multicentric study across six countries—Austria, Czechia, Poland, Hungary, Slovakia, and Slovenia—representing approximately 16% of the European Union's population. We report all pathogenic, likely pathogenic, and uncertain SOD1 variants, along with the phenotypic features, including heritability, age, site of onset, and survival. We also assessed the availability of genetic testing, counseling, and access to Tofersen therapy across the region.

Results

Out of 1200 patients with confirmed ALS, we identified 24 distinct pathogenic SOD1 variants in a total of 67 patients (median age at onset 47 [40–55] years), of whom 65.7% had familial ALS (fALS) and 34.3% had sporadic ALS (sALS). We characterized the associated phenotypes and reported that 42 patients are currently receiving Tofersen therapy.

Conclusion

This study provides the first comprehensive overview of SOD1‐ALS in Central Eastern Europe. Our findings underscore the importance of genetic testing and counseling, as well as equitable access to targeted therapies such as Tofersen to advance patient‐specific care in this region.

This multicentric study across six Central Eastern European countries—Austria, Czechia, Poland, Hungary, Slovakia, and Slovenia—identified 24 distinct pathogenic SOD1 variants in 67 patients out of 1200 with confirmed ALS, with 65.7% presenting as familial ALS. It provides the first comprehensive overview of SOD1‐ALS in the region, characterizing associated phenotypes and treatment access. Of the 67 patients identified, 42 are currently receiving Tofersen, the first EMA‐approved antisense oligonucleotide targeting SOD1 mRNA.

1. Introduction

1.1. Clinical and Genetic Background of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that primarily affects both upper and lower motor neurons in the brain and spinal cord, leading to muscle weakness, atrophy, and spasticity. As the disease progresses, patients experience difficulty speaking, swallowing, and breathing, with respiratory failure being the most common cause of death [1, 2]. ALS is a rare disorder with an incidence of approximately 2.1 in 100,000 individuals in Europe per year [3]. About 90% of ALS cases are sporadic (sALS), while 10% are familial (fALS) [4]. However, patients with sporadic ALS can also carry pathogenic variants causing the disease. Genetic mutations in genes such as SOD1, C9orf72, FUS, and TARDBP disrupt key cellular processes, leading to motor neuron degeneration. SOD1 mutations are of particular interest because they drive a toxic gain‐of‐function effect, providing the rationale for antisense oligonucleotide therapies, such as the recently approved Tofersen, which suppresses SOD1 mRNA to reduce the production of toxic SOD1 proteins [5].

Pathogenic variants in SOD1 are the second most common genetic cause of ALS in the European population, accounting for 14.8% of fALS and 1.2% of sALS. In contrast, expansions in C9orf72 are the most common, occurring in 33.7% of fALS and 5.1% of sALS [6, 7, 8]. Over 200 variants in SOD1 have been reported in the Human Gene Mutation Database [9, 10], exhibiting striking heterogeneity in clinical presentation and disease course [11], underscoring that SOD1‐ALS is not a uniform entity, but a spectrum of molecular and phenotypic subtypes. Across all ALS cases, disease onset typically occurs between 54 and 69 years of age [12], and mean survival ranges from 3 to 5 years [13].

1.2. Geographic and Phenotypic Diversity of SOD1 Pathogenic Variants

Pathogenic variants in SOD1 are not uniformly distributed geographically. In Europe, particularly in Scandinavian countries, the most prevalent variant is Asp91Ala, which typically shows a unique autosomal recessive inheritance pattern but has recently been established as causal for the dominant trait and is linked to slower disease progression and decades‐long survival [11, 14, 15, 16]. In Germany, the most prevalent variant is Arg116Gly [17], associated with fast‐progressing pure lower motor neuron symptoms and median survival below 2 years [15, 18], while in Poland it is Leu145Ser, linked with the earliest disease onset of 45 years and slowest progression (over 10 years) [11]. In North America, the most prevalent variant is Ala5Val, one of the most aggressive forms of ALS [19, 20] with rapid progression and short life expectancy [21, 22]. In contrast, His47Arg, the most prevalent variant in Asia [23, 24], is associated with a milder, slowly progressive form that primarily affects the lower limbs, a phenotype also observed in Norwegian and German carriers [23, 25, 26].

These regional differences in variant prevalence and phenotype influence diagnosis, prognosis, and therapeutic response, emphasizing the importance of genetic testing, broader population‐based studies, and their use in predictive genetic counseling.

1.2.1. Study Aim

To address the substantial data gap on SOD1‐associated ALS in Central Eastern Europe, we systematically screened for SOD1 pathogenic variants to identify individuals currently living with or newly diagnosed with SOD1‐ALS in the region. We compared genetic findings with clinical manifestations, characterizing patients within a large multicenter ALS cohort spanning six countries, together representing approximately 16.3% of the European Union population, according to Eurostat [27]. This study provides new insights into the regional heterogeneity of SOD1‐ALS and its implications for genetic counseling and targeted therapeutic approaches.

2. Methods

2.1. Study Design, Participants, and Ethical Considerations

This multicentric study was conducted across six Central Eastern European countries: Austria, Czechia, Hungary, Slovakia, Slovenia, and Poland. Participating sites in major referral centers in Innsbruck (Austria), Prague and Brno (Czechia), Ljubljana (Slovenia), and Warsaw (Poland) are members of the European Reference Networks (ERNs).

Patients were enrolled between January 2021 and June 2025. Inclusion criteria included a confirmed ALS diagnosis according to the El Escorial or Gold Coast criteria. Patients with sALS were enrolled prospectively, while those with fALS were included retrospectively to capture the full spectrum of SOD1 mutations in this region, as fALS patients were typically already diagnosed at the time of study initiation. The total number of patients enrolled per country was: Austria (n = 105), Czechia (n = 425), Hungary (n = 458), Slovakia (n = 174), Slovenia (n = 150), and Poland (n = 172). Notably, some patients screened in Czechia were of Ukrainian origin, as Ukrainians represent the largest minority group in the country.

For all centers, peripheral blood samples were collected for DNA extraction and either processed locally or sent to sequencing facilities for processing. Clinical data included sex, age at symptom onset, site of onset, disease progression tracked using ALSFRS‐R whenever possible, and survival period.

The study was approved by the Ethics Committees at participating centers: General Faculty Hospital in Prague (multicentric approval), University Hospital Motol and University Hospital Brno, Czechia; Department of Medical Genetics, Unilabs, Banská Bystrica and University Hospital Martin, Slovakia; Medical University of Warsaw, Poland; Institute of Genomic Medicine and Rare Disorders, Semmelweis University, Budapest, Hungary; Medical University of Vienna, Austria; and the Faculty of Medicine, University of Ljubljana, Slovenia. All patients provided informed consent to genetic testing.

2.2. Genetic Screening

All patients were screened for ALS‐ and motor neuron disease (MND)‐associated genes using either Sanger sequencing of the SOD1 gene (RefSeq NM_000454.5) or whole‐exome sequencing (WES) with a virtual gene panel. The panel included the four genes most commonly implicated in ALS pathogenesis: SOD1, C9orf72, TARDBP, and FUS.

For WES, genomic DNA was fragmented, libraries were prepared, and exonic regions were enriched using a hybrid‐capture exome enrichment kit. Sequencing was performed on an Illumina NovaSeq 6000 platform, and raw reads were aligned to the GRCh38 reference genome, followed by variant calling and annotation.

Identified SOD1 variants were filtered and prioritized based on population frequency data (gnomAD), in silico pathogenicity predictions (PolyPhen‐2, SIFT), and clinical evidence (ClinVar and Franklin, Qiagen). Variant classification was performed according to ACMG/AMP criteria. We included only variants classified as pathogenic, likely pathogenic, or variants of uncertain significance (VUS).

2.3. Statistical Analysis

Descriptive statistics were obtained for the entire sample. Continuous variables were reported as the median with interquartile range (IQR), and categorical variables as counts and percentages. Given the limited sample size, with some variants represented by only a single case, formal comparative statistical testing was restricted to variants with sufficient observations. Mann–Whitney U test was used for non‐parametric comparisons of continuous variables between groups. Kaplan–Meier survival curves were generated for variants with at least 5 samples to describe the survival distribution. The significance level was set at 0.05, and all tests were two‐tailed. Analyses were performed using R (version 4.6.0, R Core Team, R Foundation for Statistical Computing, Vienna, Austria).

3. Results

3.1. Cohort Description and Screening Outcome

More than 1200 patients with a confirmed ALS diagnosis in the years 2020–2025 underwent genetic screening. Among these, 67 patients (4.88%, considering index cases only) were positive for pathogenic or likely pathogenic variants in SOD1; 44 (65.7%) had fALS, and 23 (34.3%) had sALS. Notably, all patients carrying a SOD1 variant were negative for pathogenic or likely pathogenic variants in c9orf72, TARDBP, or FUS genes, supporting SOD1 as the main genetic driver of disease in this cohort. The median age at onset was 47 years (range 40–55). Two multigenerational families with ALS were included from Czechia: seven patients from family “a” and three from family “b”.

3.2. Tofersen and Riluzol Therapy

Regarding treatment, 42 patients (63% of all SOD1‐positive patients) are currently receiving Tofersen, and 54 patients (80.5%) are taking Riluzole. Patient CZ_14b was pregnant and opted to postpone Tofersen until after delivery and breastfeeding. Details on treatment status, along with clinical characteristics such as age and site of onset, motor neuron predominance, and survival rate, are shown in Table 1.

TABLE 1.

Clinical and genetic characteristics of the cohort stratified by SOD1 variant. Variant nomenclature at the cDNA (HGVS_C) and protein (HGVS_P) levels. For each patient, the age of onset (in years), ALS form (familial or sporadic), site of onset (bulbar or spinal), upper motor neuron or lower motor neuron predominance, survival period (in months), and treatment with Riluzole and Tofersen.

HGVS_C	HGVS_P	Number of cases (fALS/sALS)	Patient #	sALS/fALS	Age of onset (years)	Site of onset	LMN/UMN	Survival rate (months)	Tofersen	Riluzole
c.10 A>G	p.Lys4Glu	4/0	SK_1	fALS	68	Spinal	LMN > UMN	> 32	Y	Y
			SK_2	fALS	47	Spinal	LMN > UMN	> 120	N	N/A
			PL_3	fALS	60	Spinal	LMN > UMN	> 48	Y	Y
			PL_14	fALS	47	Spinal	LMN > UMN (left)	> 16	Y	Y
c.13 G>A	p.Ala5Thr	0/1	PL_18	sALS	45	Spinal	LMN > UMN (left)	> 12	N	Y
c.98 G>A	p.Trp33*	1/0	PL_15	fALS	43	Spinal	LMN > UMN	> 66	Y	Y
c.112 G>A	p.Gly38Arg	5–1	A_VIE5	sALS	31	Spinal	UMN > LMN(right)	> 168	Y	Y
			CZ_1	fALS	36.7*	N/A	N/A	> 200	N	N/A
			CZ_6ᵇ	fALS	34	Spinal	LMN = UMN	> 24	N	Y
			CZ_7**	fALS	41	Spinal	LMN = UMN	> 84	N	Y
			CZ_14ᵇ	fALS	28	Spinal	LMN = UMN	> 12	N	N
			CZ_15ᵇ	fALS	31	Spinal	LMN = UMN	192	N	Y
			PL_5	fALS	N/A	N/A	N/A	N/A	Y	N
			PL_7	fALS	52	Spinal	UMN > LMN	> 66	Y	Y
c.140 A>G	p.His47Arg	7/0	CZ_4ᵃ	fALS	34	Spinal	LMN > UMN (left)	> 152	Y	Y
			CZ_8ᵃ	fALS	50	Spinal	LMN > UMN (left)	> 66	Y	Y
			CZ_9ᵃ	fALS	24	Spinal	LMN > UMN (left)	214	N	Y
			CZ_10ᵃ	fALS	53	Spinal	LMN > UMN (left)	> 225	N	Y
			CZ_11ᵃ	fALS	45	Spinal	LMN > UMN (left)	360	N	Y
			CZ_12ᵃ	fALS	53	Spinal	LMN > UMN (left)	242	N	Y
			CZ_13ᵃ	fALS	36	Spinal	LMN > UMN (left)	192	N	Y
c.217 G>A	p.Gly73Ser	0/1	A_VIE7	sALS	58	Spinal	LMN > UMN (left)	> 6	Y	Y
c.272 A>C	p.Asp91Ala	1–6	A_VIE6	sALS	51	Spinal	LMN > UMN (left)	> 19	Y	Y
			CZ_5	sALS	69	Spinal	LMN > UMN (right)	† 9	N	Y
			CZ_16	sALS	57	Spinal	UMN > LMN(left)	> 8	N	N
			HU_7	sALS	43	Spinal	LMN > UMN (left)	> 72	Y	Y
			PL_13	fALS	61	Spinal	LMN > UMN	> 120	Y	N
			PL_22	sALS	54	N/A	N/A	> 42	Y	Y
			SI_3	sALS	51	Spinal	LMN > UMN (right)	> 140	Y	Y
c.304 G>A	p.Asp102Asn	1/0	PL_11	fALS	51	Spinal	LMN > UMN (left)	> 30	Y	Y
c.313 A>T	p.Ile105Phe	1/0	A_INN3	fALS	36	Spinal	UMN > LMN	> 238	Y	Y
c.319 C>G	p.Leu107Val	1/0	SK_3	fALS	47	Spinal	LMN > UMN	† 8	N	N/A
c.339 C>G	p.Ile113Met	0/1	PL_19	sALS	60	N/A	N/A	† 12	Y	Y
c.340 A>T	p.Ile114Phe	0/1	PL_10	sALS	60	Spinal	UMN = LMN	> 82	Y	Y
c.352 C>G	p.Leu118Val	2/0	HU_8	fALS	70	Spinal	UMN > LMN	> 77	Y	Y
			HU_9	fALS	44	Spinal	LMN = UMN	> 103	Y	Y
c.358 G>T	p.Val120Leu	0/1	PL_20	sALS	64	N/A	N/A	> 36	Y	Y
c.376 G>T	p.Asp126Tyr	1/0	A_INN1	fALS	47	Spinal	LMN > UMN	10	Y	Y
c.378 C>G	p.Asp126Glu	0/1	CZ_3	sALS	35	Spinal	LMN > UMN	> 216	N	N
c.412 A>G	p.Thr138Ala	1/0	A_VIE1	fALS	33	Bulbar	—	> 6	Y	Y
c.413 C>T	p.Thr138Ile	0/1	HU_1	sALS	34	Spinal	UMN > LMN(left)	† (N/A)	N	Y
c.418 A>G	p.Asn140Asp	0/1	PL_2	sALS	44	Spinal	LMN > UMN (right)	> 72	Y	Y
c.425 G>C	p.Gly142Ala	0/1	CZ_2	sALS	53	Spinal	LMN > UMN (left)	† 49	N	N
c.434 T>C	p.Leu145Ser	7–1	PL_1	fALS	65	Spinal	LMN	> 44	Y	Y
			PL_4	fALS	51	Spinal	LMN > UMN	> 64	Y	Y
			PL_6	sALS	33	Spinal	UMN = LMN	> 108	Y	Y
			PL_8	fALS	39	Spinal	LMN	> 126	Y	Y
			PL_9	fALS	33	Spinal	UMN = LMN	> 108	Y	Y
			PL_16	fALS	46	Spinal	LMN > UMN	> 54	Y	Y
			PL_17	fALS	42	Spinal	UMN = LMN	> 28	Y	Y
			PL_21	fALS	55	N/A	N/A	> 103	Y	Y
c.435 G>C	p.Leu145Phe	9–5	A_INN2	fALS	63	Spinal	LMN > UMN	> 38	Y	Y
			A_INN4	sALS	44	Spinal	LMN > UMN	> 12	N	Y
			A_INN5	fALS	66	Spinal	LMN > UMN	> 64	Y	Y
			A_VIE2	fALS	47	Spinal	LMN > UMN (left)	> 70	Y	N
			A_VIE3	fALS	49	Spinal	LMN > UMN (left)	> 18	N	N
			A_VIE4	fALS	70	Spinal	LMN > UMN (left)	> 54	N	N
			HU_2	sALS	42	Spinal	LMN > UMN (right)	> 82	N	N/A
			HU_3	sALS	34	Spinal	LMN > UMN (left)	> 50	Y	Y
			HU_4	fALS	43	Spinal	LMN > UMN (right)	> 30	Y	Y
			HU_5	fALS	62	Spinal	LMN > UMN	> 48	Y	Y
			HU_6	sALS	71	Spinal	LMN > UMN (left)	† (N/A)	N/A	N/A
			HU_10	sALS	29	Spinal	UMN > LMN	† (N/A)	N	Y
			SI_1	fALS	53	Spinal	LMN > UMN (left)	> 71	Y	Y
			SI_2	fALS	41	Spinal	LMN > UMN (left)	> 24	Y	Y
c.436 G>A	p.Ala146Thr	0/1	PL_12	sALS	53	Spinal	LMN > UMN (left)	> 16	Y	Y
c.443del	p.Gly148Valfs*2	1/0	SK_4	fALS	40	Spinal	LMN > UMN	† 10	N	Y

Note: † − deceased. The superscript letter following the patient number indicates members of the same family. * – age at inclusion, ** – patient of Ukrainian origin.

Abbreviations: A‐INN, Austria Innsbruck; A‐VIE, Austria Vienna; CZ, Czechia; HU, Hungary; fALS, familial ALS; LMN, lower motor neuron; N, no; N/A, not availablePL, Poland; sALS, sporadic ALS; SI, Slovenia; SK, Slovakia; UMN, upper motor neuron; Y, yes.

3.3. Genetic Counseling

In Austria, Slovakia, and Poland, genetic data were obtained through routine clinical genetic testing. In Czechia, SOD1 screening was performed either as part of medical genetic testing or as part of a research study. In Slovenia, genetic testing was conducted following a referral from a neurologist and a recommendation from a clinical geneticist.

Genetic counseling was generally integrated into routine interdisciplinary care for most patients with fALS. Referrals were typically initiated by neurologists, and counseling was provided in one or two sessions, conducted both before and after genetic testing. In contrast, referral patterns for genetic testing and counseling in sALS vary considerably across neuromuscular centers in Central Eastern Europe. In practice, patients with confirmed pathogenic variants are usually offered post‐test genetic counseling to discuss clinical implications, familial risk, or potential clinical trials. Although genetic testing and counseling are publicly funded and accessible to all patients with ALS, greater efforts, including patient advocacy and local guidelines, are needed to improve uptake and patient understanding.

3.4. Identified SOD1 Variants

We identified 24 distinct pathogenic or likely pathogenic SOD1 variants in the cohort. Among these, the p.Leu145Phe variant was the most prevalent, found in 14 patients (20.9%) from Austrian, Hungarian, and Slovenian origin and observed in both fALS and sALS. The most recurrent variants were p.Leu145Ser and p.Gly38Arg, each observed in 8 patients (11.94%), followed by p.His47Arg and p.Asp91Ala, each observed in 7 patients (10.45%). The p.Asp91Ala variant showed uniform geographic distribution, being detected in five of the six countries. The p.Lys4Glu variant was identified in four unrelated patients, all with fALS. The remaining variants each occurred in one or two individuals (≤ 3%), highlighting the genetic heterogeneity of SOD1‐ALS in Central Eastern Europe.

3.5. Geographic Distribution of SOD1 Variants

We mapped the regional distribution of the identified SOD1 variants across the six participating countries in Figure 1. An accompanying interactive online version provides additional details, including amino acid substitution structures, population observation, and functional annotations from the European Bioinformatics Institute (EBI) database (https://maguikhazaal.github.io/sod1‐distribution‐central‐eastern‐europe/).

FIGURE 1.

Geographical distribution of SOD1 variants across Austria, Czechia, Hungary, Slovakia, Slovenia, and Poland. Bar height represents variant frequency. The interactive version of this figure is available online at https://maguikhazaal.github.io/sod1‐distribution‐central‐eastern‐europe/.

3.6. Variant‐Specific Observations

3.6.1. p.Asp91Ala

The p.Asp91Ala variant was identified in seven heterozygote patients with ALS. This variant exhibits variable penetrance, and the mechanism of its pathogenicity in the heterozygous state has been debated. However, a recent study reported decreased neurofilament light chain (NfL) levels following Tofersen treatment, suggesting that even the monoallelic genotype may contribute to disease pathogenesis [28], although the response could also reflect an alternative or as‐yet‐unidentified molecular mechanism.

In our cohort, six of seven patients had predominantly lower‐limb onset, consistent with previous observations from a Polish study [11]. A single Czech patient had upper limb onset, a phenotype more commonly reported in Italian and German studies [14, 29]. Moreover, all but one p.Asp91Ala patient showed prolonged survival, with some exceeding 10 years. The exception was a patient with multiple comorbidities (pulmonary embolism, type 2 diabetes, chronic venous insufficiency, and a thyroid cystoid), who experienced rapid progression with a survival of only nine months, consistent with the multifactorial etiology underlying the disease.

3.6.2. p.His47Arg

In one Czech family (family a), seven patients carried this variant, with highly variable age at onset: three individuals developed symptoms early (at 24, 34, or 36 years) and four experienced later onset (at 45, 50, or 53 years). All patients presented predominantly LMN symptoms, slow disease progression, and prolonged survival exceeding 10 years, consistent with previous reports in Germany [25] and Japan [30, 31, 32].

3.6.3. p.Gly38Arg

Three relatives of a Czech family (family b) carrying this variant exhibited early onset around 31 years of age and a mixed pattern of UMN and LMN involvement, resembling the phenotype observed in the patient of Ukrainian origin (CZ_7). In contrast, an Austrian sALS patient (A_VIE5) harboring the same variant also presented early onset but displayed predominant right‐sided UMN involvement. A comparable UMN predominance was observed in a Polish patient (PL_7), who had a later onset in the fifth decade of life. One familial case (CZ_6) had a maternal history of slow‐progressing ALS with a survival of 15 years. The early onset and slow progression in our findings align with previous reports [11, 33, 34].

3.6.4. Variants Affecting Residue 145: p.Leu145Phe and p.Leu145Ser

Patients from Austria, Hungary, and Slovenia carrying the p.Leu145Phe variant showed a wide variability of age of onset, ranging from 27 to 70 years, but consistently manifested lower‐limb onset, in line with previous findings [25, 35, 36, 37]. Eight Polish patients carried the p.Leu145Ser variant, affecting the same residue, and also presented with a spinal onset.

Compared to p.Leu145Phe variant (9 fALS, five sALS), p.Leu145Ser variant occurred more frequently in familial cases (7 fALS, one sALS), and was associated with a younger median age at onset (44 [37.5–52] vs. 48 [42.25–62.75]). However, the difference in age at onset did not reach statistical significance (Mann–Whitney U test: U = 42, n ₁ = 14, n ₂ = 8, p = 0.322).

A trend towards longer survival was observed in p.Leu145Ser compared to p.Leu145Phe (median 83.5 [51.5–108] vs. 49 [28.5–65.5] months), potentially reflecting the distinct biochemical effect of the two amino acids at the protein level. Nonetheless, this observation should be interpreted with caution, as all but two patients had died by the time of the study, and longer follow‐up will be required for formal statistical analysis.

3.6.5. Variants Affecting Residue 126

We identified two different variants at position 126: p.Asp126Tyr in an Austrian patient, who died within 10 months, and p.Asp126Glu in a Czech patient who has survived for over 18 years. This contrast may reflect the radical Asp to Tyr substitution versus the conservative Asp to Glu change.

3.6.6. Variants Affecting Residue 138

We identified two different variants at position 138: p.Thr138Ala in Austria, with bulbar onset at the age of 33 years, and p.Thr138Ile in Hungary, with spinal onset at the age of 34 years. To date, p.Thr138Ala has only been described in Italian individuals with fALS [38] and sALS [39, 40], typically showing spinal symptoms with little bulbar involvement, but variability in the age of onset [38, 39]; to our knowledge, no literature reports on the p.Thr138Ile variant exist, underscoring the need for further investigation.

3.7. Survival Time

Kaplan–Meier analysis did not reveal statistically significant differences in survival across SOD1 variants (log‐rank p = 0.192) (Figure 2). The survival curve for p.His47Arg appears steeper than that of the other variants, suggesting a trend towards worse survival, which contradicts our clinical observations and the well‐established association of this variant with a longer disease course in the literature. This could be explained by small group sizes, low event rate, and high proportion of censored observations. A longer follow‐up period and a larger sample size would be required to establish genotype‐specific differences in survival across all detected variants.

FIGURE 2.

Kaplan–Meier survival curves for SOD1 variants that were present in five or more cases (p.His47Arg, p.Gly38Arg, p.Asp91Ala, p.Leu145Ser, p.Leu145Phe).

4. Discussion

The development of Tofersen has underscored the pressing need for robust data on SOD1‐ALS. Despite over three decades of research since SOD1 was first linked to ALS in 1993 [6], data from patients in Central Eastern Europe remain notably scarce. This multinational study addresses a critical gap across Austria, Czechia, Hungary, Slovakia, Slovenia, and Poland.

We included variants classified as pathogenic or likely pathogenic according to the Franklin database. We acknowledge that some variants may be considered VUS in ClinVar, reflecting differences in curation criteria; region‐specific datasets such as this may help refine pathogenicity classification and improve the clinical relevance of genetic testing in rare diseases.

Marked phenotypic heterogeneity was observed among SOD1 variant carriers, including variability in age and site of onset, progression rate, and survival. These differences could stem from distinct biochemical consequences of amino acid substitution on the protein level. SOD1 functions as a dismutase enzyme that shields cells from reactive oxygen species damage by catalyzing the conversion of superoxide radicals into oxygen and hydrogen peroxide, which are less harmful molecules. In ALS associated with SOD1 mutations, the enzyme acquires toxic gain‐of‐function properties, leading to increased oxidative stress, mitochondrial dysfunction, protein misfolding and aggregation, and disruption of cellular processes, ultimately resulting in neuronal damage.

Slower disease progression could be linked to preserved SOD1 structural stability and specific activity (e.g., p.Leu118Val) [41], despite the changes in charge and hydrophobicity (e.g., Lys4Glu, Gly38Arg) or disrupted protein interactions (e.g., p.Asp91Ala, p.Leu145Phe) [11].

Nonetheless, identifying a variant and its amino acid change alone does not reliably predict clinical course. For example, the p.Leu145Phe variant is typically associated with spinal onset [25, 35, 36, 37], yet a family of Serbian origin carrying this variant showed an unexpected respiratory onset, delaying diagnosis [42]. Another example involves an Iranian family with consanguineous parents both carrying the p.Leu145Ser variant [43], which is linked to slower disease progression [44], in whom three children developed juvenile or early‐onset ALS with a markedly aggressive course, including survival of only 2 years in two siblings and 9 years in the third, which suggests that homozygosity may drastically impact the clinical trajectory [42].

Even among patients harboring the same variant, disease expression can vary substantially. In our cohort, family members with the p.His47Arg variant exhibited uniformly slow progression but highly variable age at onset. A milder disease course with survival exceeding 20 years has been reported in Germany [25] and Japan [30, 31, 32], though the variant remains rare in Europe, with only a few reports from Germany and Norway focusing mostly on Asian or Asian‐descendant patients, highlighting the need for broader population‐based studies.

Environmental and epigenetic factors may further contribute to phenotypic variability, as illustrated by monozygotic Italian twins carrying the p.Thr138Ala variant, in whom only one developed ALS at age 35 after multiple car accidents, narcotics abuse, and head injury, while the other remained unaffected at 52 years, highlighting the potential role of non‐genetic factors [40].

To summarize genetic findings, this study examined SOD1 variants in ALS patients across six Central Eastern European countries and identified 24 unique SOD1 variants in 4.88% of the screened ALS cohort, with relatively uniform screening across the participating countries. Estimating the actual population frequency of SOD1 variants across countries would require population‐based data from all ALS patients, which was beyond the scope of this study. Population‐based registries are scarce in Central Eastern Europe, and our data help fill this gap; the findings highlight the urgency of further population‐wide efforts. We placed particular emphasis on variants affecting the same amino acid residues to assess their phenotypic impact and contribute to genotype–phenotype correlations. Familial cases revealed consistent intrafamilial profiles in some instances and considerable variability in others, underscoring the complexity of SOD1‐associated ALS. Linking identified variants to phenotypic data is a stepping stone towards integrating genetics with tailored therapies and highlights the critical role of genetic counseling in improving multidisciplinary care.

4.1. Genetic Counseling

With emerging gene‐specific treatments, genetic testing and counseling are becoming increasingly relevant for all patients with ALS, regardless of family history. Current guidelines recommend screening for at least four key genes—C9orf72, SOD1, FUS, and TARDBP—in every patient with ALS [45, 46], representing a fundamental shift from the 2012 EFNS recommendations, which advised against testing individuals without a familial history of ALS [47]. The 2024 European Academy of Neurology (EAN) guidelines support this by referring clinicians to the latest genetic counseling standards [48].

The identification of pathogenic variants in index patients raises the equally important question of when to test adult relatives and how to counsel asymptomatic carriers. Early detection helps at‐risk families make informed decisions and access early interventions, while comprehensive counseling alleviates uncertainty and offers emotional support [49].

Current testing panels range from four genes related to ALS/MND to as many as 200 genes for broader neuromuscular conditions. However, standardized protocols for presymptomatic testing remain lacking [50, 51], underscoring the urgent need to develop guidelines that balance ethical concerns with the benefits of early detection [46, 52, 53]. In Central Eastern Europe, although genetic testing and counseling are publicly funded, greater efforts are needed to increase awareness and improve patient understanding.

Moreover, as genetic testing is extended to all ALS patients, pre‐test counseling becomes an essential component of clinical care and is particularly critical for patients presenting with sALS. Unlike individuals from ALS families with known pathogenic variants, patients with sALS are often unprepared for the possibility of receiving a positive genetic result. Pre‐test counseling ensures that patients are fully informed of these potential consequences before testing is undertaken, allowing them to consider whether and how they wish to receive genetic results, and to prepare emotionally and practically for a range of outcomes, further highlighting the need for more accessible genetic counseling. This is especially relevant given that pathogenic variants are increasingly identified in ALS patients with no documented family history, as demonstrated in our cohort.

4.2. Treatment Availability

Expanding access to genetic testing and early screening is essential to ensure timely care for patients with actionable variants. Tofersen, an antisense oligonucleotide targeting SOD1, has demonstrated biological and clinical activity; integrated analyses of the phase 3 VALOR trial and its open‐label extension suggest that earlier treatment initiation is associated with slower declines in function and strength and a lower risk of death‐equivalent events, without new long‐term safety concerns [48, 54, 55], supporting its potential as a precision therapy and informing the development of other SOD1‐targeted approaches.

Riluzole was routinely prescribed as standard management, though some patients opted out due to gastrointestinal intolerance or other individual factors.

Tofersen is available under compassionate use in Czechia, Slovenia, and Slovakia through a Specific Treatment Programme (STP) or Early Access Programme (EAP), or by individual request in Hungary. Following European Medicines Agency approval, Tofersen has been accessible in Austria, funded by individual hospitals. In Poland, access was granted through an EAP, then the Rescue Access to Therapeutic Technologies (RDTL) framework, and since January 2026, it has been fully funded by the National Health Fund. Long‐term availability ultimately depends on national reimbursement policies.

5. Conclusion

This study provides a comprehensive survey of SOD1‐ALS in six Central Eastern European countries, and compares it with previously reported data, bridging a critical knowledge gap and offering insights into its genetic and clinical diversity. We investigated the effects of specific amino acid mutations and their potential correlation with clinical trajectory, though further research is needed. Assessment of neurofilament levels was not feasible at this stage but will be an essential consideration in follow‐up studies.

While genetic testing and counseling are becoming more widespread, the lack of standardized protocols underscores the need for clearer guidelines and broader access to genetic testing. Similarly, the increasing availability of Tofersen represents a significant advancement in SOD1‐ALS treatment, but ensuring health insurance coverage is essential for equitable access. Moving forward, implementing standardized genetic testing protocols and policies that facilitate access to novel therapies is crucial for improving disease management and patient care.

Author Contributions

Monika Turčanová Koprušáková: resources, writing – review and editing, investigation. Magui Khazaal: writing – original draft, writing – review and editing, visualization, methodology, data curation, formal analysis, investigation, conceptualization, project administration. Magdalena Kuźma‐Kozakiewicz: investigation, validation, resources, writing – review and editing, supervision. Adam Betík: resources, funding acquisition, writing – review and editing. Paula Stretavská: writing – review and editing, writing – original draft, investigation, resources. Eva Vlčková: resources, supervision, writing – review and editing, methodology, investigation. Krzysztof Nieporęcki: resources. Hakan Cetin: resources, writing – review and editing, investigation, supervision. Stephan Iglseder: resources. Wolfgang Löscher: writing – review and editing, resources. Blaž Koritnik: resources, writing – review and editing, investigation. Radoslav Orenčák: resources. Tamás Szlepák: resources. Christian Eggers: resources, writing – review and editing. Mária Judit Molnár: resources, funding acquisition, investigation, writing – review and editing. Daniel Baumgartner: resources. Zoltán Grosz: resources. Lenka Slachtova: funding acquisition, conceptualization, investigation, writing – original draft, methodology. Robert Petrovic: resources. Omar Keritam: resources, writing – review and editing. Magdalena Anna Koszewicz: resources.

Funding

This work was supported by Univerzita Karlova v Praze, PRIMUS UK 21/MED/012. Ministerstvo Školství, Mládeže a Tělovýchovy, OP JAK MSCA GOALS Slachtova. Fakultní nemocnice Brno, 65269705. Masarykova Univerzita, MUNI/A/1728/2025. Nemzeti Kutatási Fejlesztési és Innovációs Hivatal, TKP2021 NVA‐15, OTKA139010.

Ethics Statement

The study was approved by the Ethics Committee at each center, and written informed consent was obtained from all patients prior to their participation.

Conflicts of Interest

E.V. and A.B.: presentations focused on Tofersen treatment supported by Biogen. W.L. received speaker honoraria and consulting fees from Biogen, Roche, Anylam, Alexion, AstraZeneca, Argenx, Amicus, UCB, Janssen, and Takeda and a research grant from Pfizer. M.K.‐K. received speaker honoraria and consulting fees from Biogen, Ferrer, Cytokinetics, and Amylyx.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.