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Abstract

SYNE1 ataxia is a rare representative of autosomal recessive hereditary cerebellar ataxias (ARCAs). It was originally described in the Beauce region of Quebec as a pure cerebellar form of ataxia. Later, more complex phenotypes with a wide range of extra-cerebellar neurological and non-neurological dysfunctions were discovered in other geographical locations. SYNE1 deficiency was predominantly linked to nonsense and frameshift variants; however, missense variants in the gene SYNE1 have also been described as causative for this type of ataxia. We here describe a case of an adult female patient with a phenotype of progressive ataxic features over 15 years, combined with spastic paraparesis. Non-neurological symptoms were not present. The family history was negative, and the case was classified as sporadic. However, the parents of the patient were related to both grandmothers, being cousins. DNA was sequenced using the Illumina HiSeq/Nova Seq system, and the variant c.23413A>G; p.Arg7805Gly in the gene SYNE1 was found in a homozygous state (NM_033071.4; NP_149062.2). This missense variant has not been described in the literature and is not listed in the relevant databases. This homozygous substitution resulted in an amino acid change, arginine for glycine, in a highly conserved region of the SYNE1 gene. According to ACMG/ACGS guidelines, the criteria PM2+PM3_supporting+PP3 were applied, which classified the substitution c.23413A>G; p.Arg7805Gly as “a variant of uncertain significance.” The calculated effect of this substitution is mostly pathogenic based on several in silico prediction programs. The presence of spastic paraparesis grades this case as an ataxic complex syndrome. This is the first SYNE1 ataxia patient of Polish origin with a novel missense variant described with full clinical and MRI data.

Plain language summary

New mutation in adult patients with ataxia

The adult-onset cerebellar ataxias are rare, progressive and highly heterogenic neurological disorders, which can pose a significant diagnostic challenge. Many causes are related with these disorders. Some of them are acquired, but a large number have a genetic background. Several genes were found to be linked with ataxias. We described a new gene variant in an adult patient with ataxia. To discover genetic variant for adult onset neurological disorders is challenging and important for diagnosis, since genetic disorders manifest themselves much more frequently in early years of life. Although no treatment can be offered, knowing the cause of symptoms allow for accurate diagnosis, can help in family planning, and reduce the patient’s uncertainty. Another important contribution of the described case is demonstration that this type of genetic variant can occur in the eastern part of Europe, where it was not reported so far. Thus neurologists from this part of the world should be prepared to consider this genetic variant in diagnostic procedures.

Keywords

ARCA ataxia autosomal recessive cerebellar ataxia exome sequencing variant

Introduction

The adult-onset autosomal recessive hereditary cerebellar ataxias (ARCAs) are rare, progressive, and highly heterogenic neurologic disorders that can pose a significant diagnostic challenge. There is an extensive list of potential causative genes, and this list continues to grow, with several new ataxia genes recently identified. The 2019 consensus paper identified 59 pure ARCAs and 48 complex disorders with ataxia combined with other clinical symptoms.¹ One of the major differences between ARCA and autosomal dominant hereditary cerebellar ataxias (ADCAs) is the much less frequent genetic variation of short tandem repeats and the numerous involvement of single-nucleotide variants, insertions, deletions, and duplications.² The new causative genes involved in ARCAs have been successfully discovered thanks to advances in methods for genetic testing introduced to clinical practice in recent years.³ These genetic variants can be effectively detected with the technology of next-generation sequencing (NGS), either whole exome sequencing (WES) or whole genome sequencing.

Among several gene variants associated with ARCAs, one is related to gene SYNE1 encoding nuclear envelope spectrin repeat-1 protein (NESPRIN-1; Spinocerebellar ataxia type 8 (SCAR8); MIM* 605441; OMIM# 610743).^4,5 This protein is widely expressed in several tissues but with a strong predilection to the central nervous system and was shown to be highly expressed in the cell bodies of Purkinje cells in the cerebellar cortex and in neurons from the olivary region of the brain stem.⁶ NESPRIN-1 was localized to the postsynaptic endocytotic zone of excitatory synapses, and its knockdown disrupts glutamate receptor internalization, suggesting it may be necessary for the function of synaptic glutamate receptors.⁷ The spontaneous occurrence of recessive variants in the SYNE1 mouse spectrin beta 4 gene causes a progressive ataxia, deafness, and tremors.⁸ In 2007, variants in gene SYNE1 were described in 26 French-Canadian families from the Beauce and Bas-St-Laurent regions of Quebec as a cause of pure cerebellar ataxia.⁶ Five substitutions have been identified within the SYNE1 region, all leading to premature termination of protein synthesis. Subsequent studies discovered more complex phenotypes with a wide range of extra-cerebellar neurological and non-neurological dysfunctions associated with motor neuron and brainstem dysfunction.^4,5 In recent years, patients with a more variable clinical phenotype, including Emery-Dreifuss muscular dystrophy, arthrogryposis, and cardiomyopathy, have been reported in relation to variants in the SYNE1 gene.⁹

We report a patient with cerebellar ataxia that developed in adulthood with no family history of similar symptoms. A new SYNE1 variant was discovered in this patient with WES.

Methods

Patient recruitment and clinical data extraction

The patient was referred to the Center of Neurology in Lodz from a regional outpatient clinic in the Northern Poland and clinical data were extracted from medical documentation from previous hospitalizations and ambulatory visits as well as from direct examination at Center of Neurology and follow-up observation in the Center of Neurology for 5 years documenting slow progression of ataxia and performing several diagnostic examination, for example, MRI, nerve conduction study, and antibody testing.

WES was performed at the age of 43 years as a single exome analysis with the indication of slowly progressive cerebellar ataxia for over 15 years, combined with findings of cerebral atrophy.

Brain MRI

Brain MRI data were acquired on a 3.0 T scanner (Siemens, Erlangen, Germany). Conventional non-contrast MRI including dual-echo, repetition time (TR) 4500 ms, echo time (TE) 22 and 90 ms, 25 slices, slice thickness 3 mm, interslice gap 0.5 mm, 512 × 512 × 44 matrix, field of view (FOV) 250 mm) and T1-weighted (TR 750 ms, TE 17 ms, 25 slices, slice thickness 3 mm, interslice gap 0.5 mm, 512 × 512 × 44 matrix, FOV 250 mm), was obtained.

Sequencing

DNA was isolated from 5 mL of the patient’s peripheral blood treated with the anticoagulant EDTA. The coding and flanking intronic regions were enriched using solution hybridization technology and were sequenced using the Illumina HiSeq/Nova Seq system. CNV (copy number variations) calling was performed by computing the sample’s normalized coverage profile and its deviation from the expected coverage. At least one rare variant was re-sequenced using conventional Sanger sequencing for independent confirmation.

Computation analysis

Ilumina bcl2fast2 was used to demultiplex sequencing reads. Adapter removal was done with a skewer. The trimmed reads were mapped to the human reference genome (hg19) using the Burrows-Wheeler Aligner. The high-quality sequences were used to determine sequence variants (single-nucleotide changes and small insertions/deletions). The variants were annotated based on several databases.

Diagnostic data analysis

Variants were classified and reported based on ACMG/ACGS-2024v1.2 guidelines.¹⁰ Only variants (SNV/small indels) in the coding region and the flanking intronic regions with minor allele frequency (MAF, 1.5%) were evaluated. Known disease-causing variants were evaluated in up to ±30 bp of flanking region and up to 5% MAF. In silico prediction of variants was calculated on the basis of the output of the programs Mutation Taster, fathmm Mutation Assessor, SIFT, fathmm-MKL, LRT, and PROVEAN. In addition, the PrimateAI prediction tool was used to estimate the secondary structure and solvent accessibility based on the amino acid sequence, integrating these predictions as sub-networks within the overall model.¹¹ SpliceAI was utilized to evaluate the consequences of the variant on splicing.¹²

Results

Clinical description

The female patient of Polish origin, 43 years old, presented with an unsteady gait for the last 15 years. The symptoms were slowly progressing, leading to marked discoordination of the lower extremities and gait impairment. She reported dizziness and falls on several occasions. On neurological examination, several ataxic features have been found, such as intention tremors, dysmetria, and impaired alternating movements in the upper extremities. Similarly, ataxic symptoms were present in the lower extremities. Independent gait was maintained but was dis-coordinated and imbalanced. The muscle strength was slightly diminished with spasticity, increased deep reflexes, and the Babinski sign were found. Her speech was slurred and hesitant. No nystagmus was found (Table 1). The score of the Scale for the Assessment and Rating of Ataxia was 20/40.¹³ Cognitive, psychiatric, bulbar, and autonomic symptoms were not present, and non-neurological symptoms were not found. Nerve conduction studies and EMG were normal. The family history was negative, and no person with similar ataxic features was known to the patient. Therefore, we classified this case as sporadic. However, the parents of the patient were related to both grandmothers, being cousins, and denied further genetic testing. We excluded all potential genetic causes of cerebellar ataxia as well as other causes, including autoimmune cerebellar ataxias. Laboratory blood tests were not remarkable. CSF examination showed protein levels of 51.9 mg/mL, no oligoclonal Ig bands, no Borrelia antibodies, and no other findings related to infection. On MRI, a clear feature of cerebellum atrophy was found with vermis and cerebellar hemispheres involvement (Figure 1).

Table 1.

Clinical features.

Clinical feature	Present/no
Age of onset	28 years
Gait disturbance	Yes
Upper and lower limb ataxia	Yes
Truncal ataxia	Yes
Slow progression	Yes
Dysarthria	Yes
Intention tremor	Yes
Dysmetria and dysdiadochokinesia	Yes
Gaze-evoked nystagmus	No
Saccadic pursuit	No
Ocular dysmetria	No
Hyperreflexia	Yes
Extensor plantar responses	Yes
Sensory neuropathy	No
Cognition impairment	No

Figure 1.

Brain MRI. Sagittal (a) and axial (b) Flair sequence demonstrating atrophy of cerebellar vermis and hemispheres.

Molecular genetic testing for trinucleotide repeat expansions, SCA, 1, 2, 3, 6, 7, 17, and Friedreich ataxia (FRDA) were all negative. Single exome analysis revealed the missense variant c.23413A>G; p.Arg7805Gly in the SYNE1 gene in a homozygous state (Table 2), which is located in a spectrin repeat of the protein. In this case, 99.23% of the targeted regions were covered by a minimum of 30 high-quality sequencing reads per base. The variant was covered with 109/109 reads. This highly conserved substitution (Figure 2) has not been described in the literature and is not listed in the ClinVar database (UCSC Genome Browser; UniProt). The effect of this substitution is classified as mostly pathogenic by the in silico prediction programs used; REVEL represents with 0.64 an intermediate score, almost at the LP border. Furthermore, the detected variant is located in a splice region and might therefore have a deleterious effect on splicing, which is, however, not confirmed by the in silico splice prediction program SpliceAI, but in turn estimated as pathogenic by PrimateAI. According to ACMG/ACGS guidelines, the identified variant c.23413A>G; p.Arg7805Gly was finally classified as “variant with unknown significance (VUS)” (PM2 (moderate), PM3 (supporting), and PP3 (supporting) as arguments for low allele frequency, homozygous state, and LP/P in silico prediction). GnomAD 4.1 listed 3 heterozygotes in 1.607.062 alleles, which can be differentiated in 1 heterozygote of 812.460 females and 2 out of 801572 heterozygous males; 1 heterozygote in 1.1.79.998 non-Finnish population and 2 heterozygotes out of 91.080 alleles in the South Asian population, with a maximum frequency of 0.002%; no homozygous entries were stated. Franklin suggested using the criteria PM2 and PP3 for classifying this variant, which also resulted in VUS categorization.

Table 2.

Detection of a novel homozygous variant in gene SYNE1 (NM_033071.4).

	Gene	SYNE1
	Variant	c.23413A>G; p.Arg7805Gly
Key variant details	Genomic position (hg19)	chr6:152497530
	Transcript	ENST00000423061; NM_033071.5
	Zygosity	Homozygous
	Heredity	AR
	GnomAD	3/1.607.062
	MAF	<0.0001%
	Conservation	Highly conserved
	Coverage	109/109 reads
In silico prediction	Mutation Taster, fathmm, Mutation Assessor, SIFT, fathmm-MKL coding, LRT, and PROVEAN	Mostly pathogenic
	REVEL	Intermediate with tendency pathogenic (0,64)
	PrimateAI	Pathogenic
	SpliceAI	No splice effect

MAF, minor allele frequency.

Figure 2.

Phylogenetic conservation of the detected missense alteration over several species (excerpt from UCSC Genome Browser on Human (GRCh37/hg19)).

Discussion

The gene SYNE1, one of the largest genes in the human genome, is localized at 6q25.2, spans ~515.7 kb including introns and encodes the 8797 aa protein Nesprin-1.⁵ This protein is localized on the outer nuclear membrane and plays a major role in tethering of the nucleus to the actin cytoskeleton and maintains the structural integrity of the nucleus.¹⁴ Two N-terminal domains mediate Nesprin-1 binding to the actin cytoskeleton, whereas the C-terminal domain KASH is localized in perinucleus space and completes the nucleus and cytoskeleton complex. Nesprin-1 is localized in several tissues, and high expression was found in the cerebellum and brainstem.¹⁵ It was suggested that it plays a role in glutamate synaptic transmission.¹⁶ Variants in SYNE1 result in diseases affecting different organs, such as striated muscles, peripheral nerves, heart, and bones. Several SYNE1 variants resulted mostly in Nesprin truncated form, or frameshift reading was associated with autosomal recessive ataxia (SYNE1 ataxia), also classified as autosomal recessive cerebellar ataxia (ARCA1) or spinal cerebellar ataxia recessive (SCAR8).¹⁷ Originally described cases in the Canadian province of Beauce suggested that the clinical phenotype of this disorder is characterized by pure cerebellar ataxia and is linked to the geographic location in Canada.⁶ Later on, several other cases of SYNE1 ataxia have been described in varied regions in Europe and Asia.^4,5 It was also found that a number of patients with SYNE1 ataxia have a more complex phenotype with ataxia and additional neurological symptoms. Among them most frequent are involvement of upper and lower motor neuron disease, spastic paraparesis, and brain stem syndrome.¹⁸

In this report, we describe a new missense variant within the SYNE1 gene in a patient with cerebellar ataxia and additional features of spastic paraparesis, and widen the spectrum for missense alterations for this SCAR8. The detected homozygous c.23413A>G; p.Arg7805Gly substitution resulted in an amino acid change from arginine to glycine, in a phylogenetically highly conserved amino acid position (UCSC Genome Browser) in one of the numerous spectrin repeats of the protein (UniProt, InterPro). Arginine is a bulky, charged amino acid that has a high potential capacity for hydrogen bonds, which often plays a crucial role in maintaining a protein’s structure or in specific binding interactions.¹⁹ Replacing it with glycine, which is less affine for hydrogen bond formation, can lead to the loss of essential contacts, such as hydrogen bonds, wherethrough a destabilization of the protein could be induced.²⁰ The effect of this alteration was assessed as mostly pathogenic based on the in silico prediction programs used (analysis with >75% consensus), although the variant would not affect splicing as per in silico analysis. The c.23413A>G; p.Arg7805Gly has not been reported in literature so far, but was listed in clinical databases and was due to limited information classified as a variant of uncertain significance. The now widespread use of NGS in genetic diagnostics has generally led to an increase in the identification of variants of unclear clinical significance, underscoring the need for precise retrospective phenotyping of genetic results. In addition, further functional characterizations in future studies are necessary to clarify the effects at the protein level and to strengthen the validation of pathogenicity.

In the direct vicinity of the variant described by us, no substitution has been found in association with ataxia. Although originally SYNE1 deficiency was predominantly linked with nonsense and frameshift variants, missense changes in SYNE1 have also been described as causative for ataxia^4,21; The analysis of phenotypes in patients harboring missense variants confirmed that these patients have either pure cerebellar ataxia or ataxia combined with other neurological symptoms such as pyramidal signs, vertical gaze palsy, and cognitive impairment.^2,22–24

Clinical presentation of the case described in this report is compatible with adult ataxic cases described earlier. The female patient acquired the ataxic symptoms at the age of 30, and the disease showed relatively slow progression. The presence of spastic paraparesis classifies this case as ataxic plus syndrome. The major limitations of our findings rely on the fact that the parents’ genetic data is not available. Alternative segregation testing in the extended family was not pursued due to limited availability.

The knowledge of the prevalence of SYNE1 ataxia patients in Poland is very limited. There is one case published in a series of autosomal recessive ataxia patients, but without information on the full phenotype.²⁵ Admitting the rare prevalence of SYNE1 ataxia, these two cases support the conclusion that SYNE1 ataxia occurrence is geographically more widespread and can be found in different populations, including central/eastern Europe.

In conclusion, we first describe an individual with cerebellar ataxia in whom we detected the novel missense variant c.23413A>G; p.Arg7805Gly in the gene SYNE1. Although this variant resulted in an uncertain value, retrospective examination helps lace up the genetic landscape and marks this spot in the genetic background of SYNE1 and therefore widens the spectrum of this rare disease.

Footnotes

Acknowledgements

None.

Declarations

ORCID iD

Krzysztof Selmaj

References

Beaudin

Matilla-Duenas

Soong

, et al. The classification of autosomal recessive cerebellar ataxias: a consensus statement from the society for research on the cerebellum and ataxias task force. Cerebellum 2019; 18: 1098–1125.

Coutelier

Hammer

Stevanin

, et al. Efficacy of exome-targeted capture sequencing to detect mutations in known cerebellar ataxia genes. JAMA Neurol 2018; 75(5): 591–599.

Nemeth

Kwasniewska

Lise

, et al. Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model. Brain 2013; 136: 3106–3118.

Synofzik

Smets

Mallaret

, et al. SYNE1 ataxia is a common recessive ataxia with major non-cerebellar features: a large multi-centre study. Brain 2016; 139: 1378–1393.

Beaudin

Gamache

P-L

Gros-Louis

, et al. SYNE1 deficiency. In: GeneReviews 2018. Seattle (WA): University of Washington, 1993–2025.

Gros-Louis

Dupre

Dion

, et al. Mutations in SYNE1 lead to a newly discovered form of autosomal recessive cerebellar ataxia. Nat Med 2007; 39: 80–85.

Cottrell

Borok

Horvath

, et al. CPG2: a brain- and synapse-specific protein that regulates the endocytosis of glutamate receptors. Neuron 2004; 44: 677–690.

Parkinson

Olsson

Hallows

, et al. Mutant beta-spectrin 4 causes auditory and motor neuropathies in quivering mice. Nat Genet 2001; 29: 61–65.

Sandra

Pia

Stefano

, et al. Emery-Dreifuss muscular dystrophy type 4: a new SYNE1 mutation associated with hypertrophic cardiomyopathy masked by a perinatal distress-related spastic diplegia. Clin Case Rep 2019; 7(5): 1078–1082.

10.

Durkie

Cassidy

Berry

, et al. ACGS best practice guidelines for variant classification in rare disease. Association for Clinical Genomic Science, 2024.

11.

Sundaram

Gao

Padigepati

, et al. Predicting the clinical impact of human mutation with deep neural networks. Nat Genet 2018; 50(8): 1161–1170. (Erratum in: Nat Genet 2019; 51(2): 364.)

12.

Jaganathan

Panagiotopoulou

McRae

, et al. Predicting splicing from primary sequence with deep learning. Cell 2019; 176: 535–548.e24.

13.

Weyer

Abele

Schmitz-Hubsch

, et al. Reliability and validity of the scale for the assessment and rating of ataxia: a study in 64 Ataxia patients. Mov Disord 2007; 22: 1633–1637.

14.

Zhang

Skepper

Yang

, et al. Nesprins: a novel family of spectrin-repeat-containing proteins that localize to the nuclear membrane in multiple tissues. J Cell Sci 2001; 114: 4485–4498.

15.

Gundersen

Worman

HJ.

Nuclear positioning. Cell 2013; 152: 1376–1389.

16.

Morel

Lepicard

Rey

, et al. Drosophila Nesprin-1 controls glutamate receptor density at neuromuscular junctions. Mol Life Sci 2014; 71(17): 3363–3379.

17.

Wiethoff

Hersheson

Bettencourt

, et al. Heterogeneity in clinical features and disease severity in ataxia-associated SYNE1 mutations. J Neurol 2016; 263: 1503–1510.

18.

Fratto

Morelli

Buonocore

, et al. Hereditary spastic paraplegia associated with ultra-rare variant enrichment encompassing the SYNE1, CAPN1 and PGAP1 genes. Cerebellum 2024; 24(1): 3.

19.

Hristova

Wimley

WC.

A look at arginine in membranes. J Membr Biol 2011; 239(1–2): 49–56.

20.

Dong

Sharma

Zhou

H-X

, et al. Glycines: role in α-helical membrane protein structures and a potential indicator of native conformation. Biochemistry 2012; 51(24): 4779–4789.

21.

Mademan

Harmuth

Giordano

, et al. Multisystemic SYNE1 ataxia: confirming the high frequency and extending the mutational and phenotypic spectrum. Brain 2016; 139: 1–6.

22.

Arias

Mir

Fernández-Matarrubia

, et al. Autosomal recessive spinocerebellar ataxia SCAR8/ARCA1: first families detected in Spain. Neurologia (Engl Ed) 2022; 37(4): 257–262.

23.

Ngo

Rexach

Lee

, et al. A diagnostic ceiling for exome sequencing in cerebellar ataxia and related neurological disorders. Hum Mutat 2020; 41(2): 487–501.

24.

Duan

Hao

Cao

, et al. Autosomal recessive cerebellar ataxia type 1: phenotypic and genetic correlation in a cohort of Chinese patients with SYNE1 variants. Cerebellum 2021; 20(1): 74–82.

25.

Krygier

Kwarciany

Wasilewska

, et al. A study in a Polish ataxia cohort indicates genetic heterogeneity and points to MTCL1 as a novel candidate gene. Clin Genet 2019; 95(3): 415–419.

Novel homozygous SYNE1 missense variant in late onset autosomal recessive cerebellar ataxia 1: a case report