Dystonia-ataxia syndrome with permanent torsional nystagmus caused by ECHS1 deficiency.

Abstract Biallelic mutations in ECHS1, encoding the mitochondrial enoyl‐CoA hydratase, have been associated with mitochondrial encephalopathies with basal ganglia involvement. Here, we describe a novel clinical presentation consisting of dystonia‐ataxia syndrome with hearing loss and a peculiar torsional nystagmus observed in two adult siblings. The presence of a 0.9‐ppm peak at MR spectroscopy analysis suggested the accumulation of branched‐chain amino acids. Exome sequencing in index probands identified two ECHS1 mutations, one of which was novel (p.V82L). ECHS1 protein levels and residual activities were reduced in patients’ fibroblasts. This paper expands the phenotypic spectrum observed in patients with impaired valine catabolism.


Neuroimaging
We performed Magnetic Resonance Imaging (MRI) using a 3 tesla scanner (Philips Achieva), acquiring morphological T1 and T2 images with turbo field echo (TFE) and fluid attenuated inversion recovery (FLAIR) 3D sequences, turbo spin echo (TSE) 2D sequences, completed with spectroscopy studies (MRS) using a single voxel placement in basal ganglia with a point RESolved spectroscopy sequence (PRESS) with a TE of 144 e 35 ms each.

Genetic Studies
Exome libraries were enriched using the SureSelect CRE v2 Kit (Agilent) and sequenced on a NextSeq550 instrument. FASTQ files were aligned to the human GRCh37.p11 genome build. Variants were prioritized by using Enlis genome software (https://www.enlis.com) and manually inspected by using IGV (https://software.broadinstitute.org/software/igv/). ECHS1 mutations were confirmed at genomic and cDNA (obtained from skin fibroblasts RNA) level by Sanger sequencing. Supplementary Figure 1 provides additional description of NGS metrics and variants prioritization workflow.

Cellular studies
Skin fibroblasts were cultured in DMEM supplemented with 15% FBS. Neuroblastoma adherent cell line SH-SY5Y were maintained in DMEM/F12 nutrient mixture supplemented with 10% FBS, 1% Penicillin-Streptomycin, 2.5 mM L-Glutamine and 1x NEM NEAA. Immunocytochemistry (ICC) analysis of ECHS1 was performed as previously described [S2]. JC1 staining (Sigma CS0390) was performed according manufacturer instruction. Oil-Red O (ORO) staining was performed as described [S3] with little modification (reduction of washing steps time and incubation with ORO reagent for 1 hour). ECHS1-silenced SH-SY5Y cells were obtained by using Lipofectamine® RNAi-MAX Transfection Protocol (Thermo Fisher Scientific, Invitrogen #13778075) to transfect TriFECTa® RNAi Kit (DsiRNAs: ECHS1-2 and ECHS1-3, IDT). Different conditions (single/double siRNA transfection, time of treatment, concentration of siRNA) were evaluated to find the optimal conditions for silencing of targets, preserving the recommended ratio between Lipofectamine e siRNA. 24 hours of co-administration of 30 nM ECHS1-2 and ECHS1-3 sequences resulted in a 70% transcript reduction without a significant increase in cellular mortality. L-Valine (Sigma-Aldrich, Lot #SLBS4325) was added to culture medium at 5 mM and 50 mM final concentrations for variable times.

Statistical Analysis
Results from fibroblasts of the two patients were compared to three to five age matched-control subjects, according the experiment. The number of replicates is indicated in the figure legends. A two-tailed Student's t test was used for statistical analyses. Graphs in figures represent mean ± s.d. with a statistical significance of * * p < 0.05.

SUPPLEMENTARY FIGURE 9
Oil Red O Solution staining in control and patients' fibroblasts. Lipid accumulation (red dots) was not detected in cells cultured in standard condition or after the addition of increasing concentration of L-Valine (5 mM and 50 mM, 24 hours), regardless of their genotype.

SUPPLEMENTARY FIGURE 10
The chart shows ECHS1-mutated subjects for those the quantification of residual enzyme activity in fibroblasts (crotonyl-coA used as substrate) is available. Y-axis indicate the percentage of residual activity compared to controls used in the relative study. X-axis shows the maximum age reached, as reported. Red color indicates dead subjects. Green color indicates patients who were alive at the moment of their description. Pearson's correlation test indicates a positive (r = 0.88) and significant (p<0.05) correlation. Abbreviations: d (days), m (months), y (years).