Clinical Phenotype Comparison in Polish Patient Cohorts with and Without Molecular Diagnosis of Dystonia

Abstract

Background: Dystonia is a heterogeneous hyperkinetic movement disorder characterized by sustained or intermittent muscle contractions causing abnormal movements and postures. Although numerous genes associated with dystonia have been identified, the genetic background remains unknown in many patients. Data on genotype–phenotype correlations in Polish populations remain limited. Objective: To analyze the clinical characteristics of patients with generalized dystonia and compare clinical features between individuals with and without genetically confirmed dystonia-causative variants in a Polish cohort. Methods: A retrospective analysis of patients diagnosed with generalized dystonia at a single neurological center was performed. Diagnosis was established according to MDS criteria. Genetic analysis included whole-exome sequencing, targeted NGS genetic panel, MLPA, Sanger sequencing and PCR_RFLP analysis. Clinical and demographic data were extracted from medical records. Clinical characteristics of individuals with and without causative variants were compared. Results: A total of 113 patients with generalized dystonia were included. Genetic variants were identified in 13 patients (11.5%). These included variants within the TOR1A, THAP1, SGCE, GCH1, NKX2-1, SLC2A1, KMT2B, PDHA1, MFN2, and GNAL genes. We found detailed clinical data of 46 patients included in the study. Our comparative analysis of patients with causative (n = 7) and without causative variants (n = 39) revealed no statistically significant differences in age of onset, initial symptom localization, treatment response, family history, or associated neurological features. Conclusions: In this cohort of Polish patients with generalized dystonia, we identified pathogenic variants in approximately 11.5% of cases. No significant clinical differences were observed between patients with genetically confirmed dystonia and those without identified variants. In this study, we report the first two Polish cases with DYT-GNAL variants. Further studies are required to reveal the clinical heterogeneity of dystonia and characterize dystonia subtypes.

1. Introduction

Dystonia can be characterized as both a symptom that is part of a broader clinical picture of certain neurological conditions, and a disease in itself—a hyperkinetic movement disorder in which sustained or repetitive muscle contractions occur involuntarily. A revised classification of dystonia categorizes this condition based on several features, such as body distribution, age at onset, progression pattern, and accompanying symptoms [1]. Body distribution can be focal, segmental, or generalized. Focal dystonia is defined as symptoms limited to one body area. Segmental dystonia manifests in two neighboring regions, whereas generalized dystonia affects the whole body [1,2,3]. Nowadays, novel genetic techniques are applied to discover new genetic factors associated with dystonia occurrence [4]. A genetic background is usually identified in generalized dystonia, particularly in cases with symptom onset before 18 years of age and a positive family history [5]. If the only symptoms are dystonia, the condition is classified as isolated dystonia, whereas if other comorbid symptoms are present, it is referred to as combined dystonia [1,2].

Recently, thanks to the ongoing advancement of molecular genetics’, numerous causative variants have been identified. Approximately 400 have been associated with dystonia According to the OMIM database, 38 genes are related to monogenic forms (OMIM; Phenotypic Series—PS128100, omim.org/phenotypicSeries/PS128100); however, in most cases, a genetic underlying cause remains unidentified. As in some cases, the response to certain treatments may vary depending on the patients ‘genetic status [6]. Therefore, there is a strong need to determine this status in new populations. There is limited data on cross-sectional cohort analyses in Europe. However, we found several reports from large cohort studies. A German paper included a large cohort from their national DysTract registry, which revealed 137 distinct likely pathogenic/pathogenic variants. The authors concluded that generalized dystonia and an age of onset <30 years old were the strongest predictors of an underlying genetic basis of the disease [7]. Other studies focused on the specific type of dystonia or gene responsible for the disease, such as the GCH1 variant in the Spanish population or cervical dystonia and blepharospasm in the Hungarian population [8,9]. To date, studies including the Polish population were mostly limited to case reports or single gene analyses, which highlights the need for future larger cohort investigations [10,11,12,13,14,15,16].

The aim of this study was to conduct a single-center analysis of patients with generalized dystonia and correlate their clinical symptoms with whether they possessed known variants in genes associated with dystonia.

2. Materials and Methods

2.1. Clinical Analysis

The MDS diagnostic criteria were applied by experienced movement disorder specialists for the diagnosis of generalized dystonia (D.K., M.F., S.S. and L.M.) [1,2]. Clinical and demographic data were collected retrospectively using past medical records from the Department of Neurology, Faculty of Health Science, Medical University of Warsaw. The study received approval from the Bioethics Committee of the Medical University of Warsaw and was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki (KB/56/2018). Most of the patients were initially screened for DYT-TOR1A variants, as this represents the most common genetic cause of generalized dystonia [17]. Further targeted genetic analyses were performed when specific clinical features were present, for example, myoclonus suggestive of an SGCE variants or thyroid dysfunction suggestive of an NKX2-1 variants.

2.2. Genetic Analysis

Genetic analysis was performed in all patients included in the study. Molecular analyses were performed on gDNA isolated from peripheral blood leukocytes using standard procedures. Different molecular techniques were implemented, depending on clinical referral (Supplementary Table S1).

Different molecular techniques were implemented, depending on clinical referral.

In regard to the most common DYT1, the analysis included the presence of ΔGAG DYT1 deletion in the TOR1A gene using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). This analysis was performed in 103 patients. Sanger sequencing as a singular method was used in 44 patients. It included the following genes: GCH1 (2 patients), THAP1 (28 patients), SGCE (17 patients), SLC2A1 (8 patients), PNKD (2 patients), PRRT2 (4 patients) NKX2-1 (1 patient), TH (1 patient) and SPR (1 patient). MLPA as a singular method was performed in 23 patients. The MRC Holland Salsa P099, P059 and P138 (Amsterdam, The Netherlands) was used for analysis, including GCH1, TH, SGCE, PRRT2 (P099; 16 patients), TOR1A, THAP1, ATP1A3, and PRKRA (P059; 9 patients), SLC2A1 and STXBP1 (P138; 4 patients) gene exon dosage probes.

The targeted NGS sequencing panel was performed in 4 patients. The panel which included the following genes was analyzed: ADAR, ADCY5, ANO3, ATM, ATP1A3, ATP7B, BCAP31, CACNA1A, COASY, COL6A3, COX20, DNAJC12, FA2H, FBXO7, FTL, GCDH, GCH1, GNAL, HPCA, KCD17, KCNA1, KCNMA1, KIF1C, KMT2B, MECR, PANK2, PINK1, PLA2G6, PNKD, PRKN, PRKRA, PRRT2, SCN8A, SGCE, SLC19A3, SLC2A1, SLC30A10, SLC39A14, SLC6A3, SPR, TAF1, TH, THAP1, TOR1A, TUBB4A, VAC14, VPS13A. Targeted genes were enriched using KAPA Hyper Choice and sequencing was then performed using the MiSeq platform (Illumina, San Diego, CA, USA).

WES was performed in 2 patients. The DNA library was prepared using the Human Exome 2.0 Plus Comprehensive Exome Spike-in (TwistBioScience, South San Francisco, CA, USA) and sequencing was performed on NovaSeq6000 platform (Illumina, USA).

Sequences were mapped and compared to the human reference genome version GRCh38/hg38.

Sequencing data were analyzed using a robust end-to-end in-house pipeline “IMC_pipeline” (data on request), with annotations of variants VEP (Ensembl release 114).

The presence of all variants identified by NGS was later confirmed with target Sanger sequencing.

The identified variants were referred to canonical reference sequences GRCh38 NM_ MANE Select [18] and described according to HGVS v.21.0.4 recommendations [19].

The most identified variants have been previously documented in the Human Gene Mutation Database Professional [20] and/or ClinVar database [21].

The pathogenicity of identified variants was classified according to American College of Medical Genetics and Genomics standards and guidelines [22].

2.3. Statistical Analysis

The statistical analyses were conducted using STATISTICA v.13.5 software, TIBCO, Palo Alto, CA, USA. The normality of distribution was evaluated with the Shapiro–Wilk test. Continuous variables were presented as means and standard deviations. Categorical variables were presented as frequencies (percentages). Parametric data were compared using the independent t-test, and the categorical data were compared with Fisher’s exact test (two-sided).

3. Results

A total number of 113 patients (44.2% males (n = 50)) were included in the analysis, which revealed 13 with causative variants (11.5%)-3 TOR1A (2.7%), 1 THAP1 (0.9%), 1 SLC2A1 (0.9%), 1 NKX2-1 (0.9%), 1 GCH1 (0.9%), 2 SGCE (1.8%), 2 GNAL1 (1.8%), 1 PDHA1 (0.9%) and 1 MFN2 (0.9%) in 1 patient and 1 KMT2B (0.9%) (

Table 1. Table with variants discovered in analyzed cohort.

Patient	Sex	Gene Name	Variant		CADD Phred	Mode of Inheritance	Zygosity	Classification	Previously Reported	Reference Sequences
			c.DNA	Protein
P1	female	TOR1A	c.907_909delGAG	p.Glu303del	19.72	AD	heterozygous	P	Yes/ClinVar RCV000005488.8; HGMD 2026.1 CD972174	NM_000113.3
P2	female	TOR1A	c.907_909delGAG	p.Glu303del	19.72	AD	heterozygous	P	Yes/ClinVar RCV000005488.8 HGMD 2026.1 CD972174	NM_000113.3
P3	female	TOR1A	c.907_909delGAG	p.Glu303del	19.72	AD	heterozygous	P	Yes/ClinVar RCV000005488.8 HGMD 2026.1 CD972174	NM_000113.3
P4	male	THAP1	c.15C>G	p.Cys5Trp	23.40	AD	heterozygous	LP	Yes/VCV000807708.4 HGMD CM149026	NM_018105.3
P5	male	NKX2-1	c.348del	Cys117Alafs*8	-	AD	heterozygous	LP	No	NM_001079668.3
P6	female	SGCE	c.1310C>T	p.Pro437Leu	26.80	AD	heterozygous	VUS	Yes/ClinVar VCV000373239.5	NM_003919.3
P7	female	SGCE	c.742T>C	p.Cys248Arg	27.70	AD	heterozygous	LP	No	NM_003919.3
P8	male	SLC2A1	c.652C>T	p.Arg218Cys	25.70	AD	heterozygous	VUS	Yes/ClinVar VCV000095415.19; HGMD 2026.1 CM2318414	NM_006516.4
P9	female	GNAL	c.385 G>A	p.Glu129Lys	35.00	AD	heterozygous	VUS	No	NM_001142339.3
P10	female	GNAL	ex1-12del #	p.?		AD	heterozygous	P	Yes/ClinVar VCV000148429.2, VCV000154072.3, VCV002579191.1; HGMD 2026.1 CG177362, CG1413517, CG2312382	NM_001142339.3
P10	female	AFG3L2	ex10-17del ##	p.?		AD	heterozygous	P
P11	female	GCH1	c.453+1G>A	p.?	32.00	AD	heterozygous	P	Yes/ClinVar VCV002925519.3; HGMD 2026.1 CS093875	NM_000161.3
P12	male	MFN2	c.2119C>T	p. Arg707Trp	29.30	AD	heterozygous	P/LP	Yes/ClinVar VCV000002280.80; HGMD 2026.1 CM081695	NM_014874.4
P12	male	PDHA1	c.784 G>C	p.Val262Leu	25.50	XLD	hemizygous	P	Yes/ClinVar VCV004070370.1; HGMD 2026.1 CM2213231	NM_000284.4
P13	female	KMT2B	c.2885G>A	p.Arg962His	28.90	AD	heterozygous	LP	Yes/ClinVar VCV002498773.24	NM_014727.3

^# NC_000018.10:g.[(?_11689564)_(11881135_?)del];[=], ^## NC_000018.10:g.[(?_12377082)_(12329565 _?)del];[=], AD—autosomal dominant, LP—likely pathogenic, P—pathogenic, XLD—X-linked dominant.

). Due to the retrospective nature of the study, only 46 patients possessed detailed clinical data, 7 of which had confirmed causative variants. The age of onset was similar in patients with and without causative variants. There were no differences in the body region of first symptom onset. There were no differences in the response to any treatment. The detailed results can be found in

Table 2. Comparison of the clinical data of patients withs (n = 7) and without (n = 39) variants associated with dystonia.

	Patients with Dystonia-Associated Variants (n = 7)	Patients Without Dystonia-Associated Variants (n = 39)	p Value
Age of onset	19.0 ± 12.7	25.4 ± 24.2	0.40
First symptom: lower limb onset	0	13 (27.3%)	0.16
First symptom: trunk onset	1 (14.2%)	7 (16.73%)	1
First symptom: upper limb onset	2 (28.6%)	25 (64.1%)	0.09
First symptom: head and neck onset	3 (42.9%)	25 (64.1%)	1
Positive family history	3 (42.9%)	14 (35.9%)	0.65
Response to any treatment	1 (14.2%)	23 (69.7%)	0.06
Myoclonus presence	2 (28.6%)	8 (20.5%)	0.63
MRI changes	3 (42.9%)	13 (33.3%)	0.4
Chorea presence	1 (14.2%)	1 (2.6%)	0.29
Thyroid insufficiency	2 (28.6%)	1 (2.6%)	0.28

.

4. Discussion

Generalized dystonia remains one of the most disabling and burdensome movement disorder [23]. Identifying the genetic background is clinically important, as some forms of dystonia may have different responses to various therapeutic approaches depending on the underlying variant. For example, DYT-KMT2B and DYT-TOR1A dystonias have been reported to respond particularly well to deep brain stimulation (DBS), showing the importance of genetic diagnoses in clinical decision-making [24,25]. In our cohort, we found one patient with KMT2B and three patients with DYT-TOR1A. One female DYT-TOR1A subject responded very well to bilateral DBS GPi.

In our cohort, we cautiously observed that establishing a specific genetic diagnosis based solely on clinical symptoms of generalized dystonia may be difficult. Although some distinct clinical features may be associated with specific genetic forms, their clinical presentation is often highly heterogeneous. For example, thyroid dysfunction and pulmonary involvement have been reported in NKX2-1-related dystonia, whereas GCH1-related dystonia typically presents before the age of 18 years with lower limb dystonia and a good response to levodopa treatment [26,27,28]. Additionally, some forms of dystonia associated with neurodegeneration related to brain iron accumulation or Wilson disease may present with characteristic neuroimaging features [29].

Our study analyzed a population that has been underrepresented in genetic studies of dystonia. The most interesting findings in our report are two cases possessing the GNAL variant. Interestingly it was responsible for almost 2% of all generalized dystonia in our cohort. In the first family, one patient presented with symptoms involving the neck and limbs, with symptom onset at the age of 14. The p.Glu129Lys variant was not previously reported and had no functional studies. However, it has a high CADD score could be potentially responsible for the patient’s dystonia. The second family possessed the heterozygous GNAL deletion with a concomitant AFGL32 deletion reported in the proband. The symptoms initially began in the face and neck at 18 years of age. The patient underwent DBS GPI at 37 years of age with no clinical response. Notably, the patient had no significant family history. However, the NGS panel study is not a method of choice to confirm the copy number variants and requires additional confirmation in methods like MLPA or aCGH [30].

GNAL variants have previously been associated with primary dystonia and almost 100 patients have been reported to date [31,32]. The inheritance pattern is typically autosomal dominant with limited penetrance in affected families [33,34]. Symptom onset usually occurs from early to late adulthood, with a median age of onset of around 38 years. In most patients, symptoms begin in the neck and craniocervical region and may progress to segmental, multifocal, or occasionally generalized dystonia. The GNAL gene is located on the short arm of chromosome 18 (18p11.21) Additional clinical features can include spinocerebellar ataxia, facio-scapulo-humeral muscular dystrophy, and dystonia, with GNAL deletion considered the main driver of dystonia in this syndrome. Treatment is usually limited, but there have been reports of 15 cases of individuals who underwent DBS surgery, mostly revealing improvement [32]. The second patient has a concomitant deletion of the AFGL32 gene. The variant in this gene is associated with several diseases like spinocerebellar ataxia type 28, optic nerve atrophy, spastic ataxia type 5 and dystonia parkinsonism. AFG3L2 interacts with OPA1, which encodes a mitochondrial GTPase responsible for the fusion of the inner mitochondrial membrane [35].

SLC2A1 variants are associated with glucose transporter type 1 deficiency syndrome (GLUT1-DS). Seizures are the most reported symptom, followed by movement disorders such as paroxysmal exertional dyskinesia, dystonia, and ataxia. A ketogenic diet is the first-line treatment [36]. A heterozygous SLC2A1 p.Arg218Cys variant was identified in a patient whose symptoms began at 22 years of age. The initial presentation was cervical dystonia, which later progressed to more generalized symptoms. A ketogenic diet was recommended; however, due to loss to follow-up, the treatment outcome remains unknown.

We also identified two new patients with SGCE variants in our cohort, which is the most common genetic cause of myoclonus-dystonia. In Poland, the SGCE variants has previously been reported in two families [14,15].

We also found a patient with levodopa-responsive dystonia. Variants in GCH1 are responsible for dopa-responsive dystonia and are typically associated with early onset and a good response to levodopa treatment [36]. In Poland, only three cases of GCH1 variants have been previously reported. Interestingly, the variant identified in our study had also been reported in a 15-year-old Italian patient presenting with gait disturbances [37].

We also included into our study three patients previously reported on in the literature with DYT-TOR1A variants [13]. DYT-TOR1A was the first genetically described form of dystonia and typically symptoms manifest before the age of 18 [38]. Previous studies estimated the prevalence of the TOR1A variant in patients with dystonia to be approximately 6% [39]. However, only two previous studies have analyzed TOR1A variants in Polish patients with dystonia. Those reports showed a relatively high prevalence (20.8% and 7%), which is higher than that observed in our cohort (2.7%) [11,13]. This discrepancy may be associated with smaller sample sizes in previous studies as well as differences in patient selection. In addition, our cohort only included patients with generalized dystonia.

The previously reported patient had two confirmed variants: an X-linked PDHA1 variant (causing pyruvate dehydrogenase complex deficiency) and an autosomal dominant MFN2 variant responsible for Charcot–Marie–Tooth disease type 2A2A or hereditary motor and sensory neuropathy type VIA16. The patient’s first symptoms appeared at 8 years of age, which is unusual for pyruvate dehydrogenase complex deficiency, as the disease typically presents in infancy and only a few patients survive into late childhood. The coexistence of the second mutation may have contributed to the unique phenotype of generalized dystonia [16]. Patients with confirmed variants included in this study were also reported in other studies: NKX2-1 [26,40], PDHA1 and MFN2 [16], THAP1 [12].

Several limitations of this study should be acknowledged. Firstly, the retrospective design resulted in incomplete clinical data for some patients, and genetic testing approaches were not uniform across the cohort. Additionally, certain variants identified through NGS-based panels—such as copy number variations (CNVs) and exonic rearrangements—require confirmation using complementary methods, including array comparative genomic hybridization. The pathogenicity of variants of uncertain significance require further investigation in a functional study setting. Moreover, the relatively small sample size in the comparative analysis limits the statistical power of the study. As a result, the lack of statistically significant differences between patients with and without causative variants should be interpreted with caution. Despite these limitations, this study represents the first analysis of this kind in the Polish population. Future research involving larger cohorts and more comprehensive genetic testing strategies may enable more precise genotype–phenotype correlations.

5. Conclusions

Our study presents an overview of the genetic background of generalized dystonia in a Polish cohort. Pathogenic variants were identified in several dystonia-associated genes; however, no clear differences in clinical presentation were observed between patients with and without dystonia causative variants. These findings underscore the clinical heterogeneity of generalized dystonia and highlight the importance of genetic testing for accurate diagnosis. Furthermore, genetic analysis should be routinely considered in patients with generalized dystonia, as clinical features alone may not reliably predict the underlying etiology.