A 53-year-old Italian man was referred to our hospital because of a progressive gait disturbance and difficulty in daily living activities.

He was born to non-consanguineous parents after an uneventful gestation and delivery. Family history was unremarkable. His sister reported that he had always had learning difficulties, but no further information could be obtained about his development. Since childhood, involuntary, irregular, and arrhythmic movements were observed in the upper limbs, that did not improve following alcohol consumption. Additionally, the patient denies experiencing any seizures.

The patient presented with an ataxic broad-based gait and an inability to walk in tandem. There was moderately dysarthric speech. The patient exhibited oculomotor disturbances: square wave jerks’ saccadic intrusion, fragmented and slow pursuit movements, and “round the house” sign in vertical saccadic movements. Involuntary, jerky, high-frequency movements were observed in the upper limbs. The patient exhibited an action tremor that worsened reaching the target (Supplementary Material 1 - Videos). On the Scale for the Assessment and Rating of Ataxia (SARA), he obtained 9/40 points. A neuropsychological evaluation demonstrated mild to moderate intellectual disability [Mini-Mental state examination (MMSE): 19.9; Montreal Cognitive Assessment (MoCA): 17/30; total IQ evaluated through Wechsler Adult Intelligence Scale – IV: 52 (Verbal Comprehension Index = 69, perceptual Organization Index = 6, working Memory Index = 66, processing Speed Index = 53)].

Brain MRI was normal, except for a slight widening of the subarachnoid spaces (Fig. 1). An electroencephalogram (EEG) showed bilateral anterior occasional slow waves with angular appearance, without clinical correlation. A surface electromyography (EMG) examination showed that the upper limbs tremolous movement was caused by frequent and arrhythmic short EMG discharges indicative of myoclonus. The cortical origin of myoclonus was revealed with jerk-locked back-averaging and cortico-muscular coherence analysis (Fig. 2). EEG-EMG analysis was performed with Brainstorm [5], which is documented and freely available for download online under the GNU general public license (http://neuroimage.usc.edu/brainstorm).

Brain magnetic resonance imaging (MRI): (A) T1-weighted axial image and (B) T1-weighted sagittal image show a slight widening of the subarachnoid spaces of the cranial vault (grey arrows) without evidence of cerebellar or brainstem atrophy

Neurophysiological assessment: (A) Electromyographic (EMG) trace: EMG1 + = Right wrist extensor, EMG2 + = Right wrist flexor recorded with the arms outstretched in front of the chest. The EMG trace shows frequent bursts of very short duration (30 ms) on EMG1+, which are often observed synchronously on the antagonist muscle EMG2+, compatible with myoclonus. The number of bursts per second is irregular and varies during the recording; (B) Jerk-Locked Back Averaging (JLBA): the JLBA performed on 71 EMG discharges at about 30 milliseconds shows a positive-negative wave on the contralateral central area (C3), with maximum positivity at -15 milliseconds from the onset of the myoclonus (time 0). No recognizable waves are seen on C4; (C) the averaging of the 71 myoclonic jerks, visible synchronously on EMG1 + and, with lower amplitude, also on EMG2+; (D) a voltage map shows the temporal trend of EEG voltages at -40, -26, -14, and 0 ms from the myoclonus. Red, white, and blue colors on the voltage map indicate respectively positive, isoelectric, and negative voltage. The highest positive voltage can be observed at -16 milliseconds over C3 area, that turns into a negative voltage at 0 milliseconds. (E) Cortico-muscular coherence analysis shows high peaks of coherence within the beta frequencies band (from 16 to 29 Hz) with the highest peak on 22–23 Hz. The horizontal black line indicates the 95% significant coherence level, set at 0.055. EEG–EMG data analysis was performed with Brainstorm which is documented and freely available for download online under the GNU general public license (http://neuroimage.usc.edu/brainstorm)

Metabolic [plasma lactate, amino acids, acylcarnitine in dried blood spot (DBS), guanidinoacetate (DBS), urinary Creatine/Creatinine ratio, urinary organic acids, urinary pterins, ceruloplasmin, plasma and 24 h urinary copper, alpha fetoprotein] and genetic studies (SNP-array, karyotype, FMR1 pre-mutation test, and pathological expansions in polyglutamine SCA1-2-3-6-7-8-12-17) were all normal.

Massive, targeted sequencing with a panel of 285 genes (Supplementary Material 2 - Supplementary Informations) known to cause hereditary ataxias or complex syndromes in which ataxia is a symptom [6] showed a novel c.3835G > A (p. Asp1279Asn) variant in CACNA1G. The variant was ultrarare (gnomAD frequency 0.0001%) and predicted to be damaging in silico by all algorithms we tested (CADD score 31, REVEL 0.74, AlphaMissense 0.93); it was categorized as class 4/Likely pathogenic according to Varsome (https://varsome.com/, accessed on March 2024) (criteria PS4, PM2, PP3 following the ACMG guidelines [7]). In silico analysis of experimentally determined protein structure (8) showed that mutated residue lies in the cytoplasmic side of segment 1-repeat III (S1III), hence being potentially involved in voltage-sensing function (Fig. 3).

In silico analysis of mutated residue. (A) Assessment of amino acid localization was performed with Protein Data Bank of Transmembrane Proteins (PDBTM) using an experimentally determined protein structure (PDB entry: 6KZO) and revealed that aspartate in position 1279 is placed in the cytoplasmic side of S1III domain. (B) Representation of wild-type and mutant residues within protein structure. Dotted orange line = polar bond, dotted light blue line = van der Waals forces, grey spheres = carbon atoms, red spheres = oxygen atoms, blue spheres = nitrogen atoms, yellow spheres = sulfur atoms

The patient was started on zonisamide 50 mg, which resulted in minimal improvement in the myoclonus and no benefit on ataxia. The addition of clonazepam (2 mg/day) resulted in a significant improvement in the myoclonus.