https://doi.org/10.1093/braincomms/fcac030 BRAIN COMMUNICATIONS 2022: Page 1 of 21 | 1 BRAIN AIN COMMUNICATIONS New spinocerebellar ataxia subtype caused by SAMD9L mutation triggering mitochondrial dysregulation (SCA49) Marc Corral-Juan,1 Pilar Casquero,2 Natalia Giraldo-Restrepo,2 Steve Laurie,3 Alicia Martinez-Piñeiro,4 Raidili Cristina Mateo-Montero,2 Lourdes Ispierto,5 Dolores Vilas,5,6 Eduardo Tolosa,6 Victor Volpini,7 Ramiro Alvarez-Ramo,5 Ivelisse Sánchez1 and Antoni Matilla-Dueñas1 Spinocerebellar ataxias consist of a highly heterogeneous group of inherited movement disorders clinically characterized by progres- sive cerebellar ataxia variably associated with additional distinctive clinical signs. The genetic heterogeneity is evidenced by the myriad of associated genes and underlying genetic defects identified. In this study, we describe a new spinocerebellar ataxia subtype in nine members of a Spanish five-generation family from Menorca with affected individuals variably presenting with ataxia, nystagmus, dys- arthria, polyneuropathy, pyramidal signs, cerebellar atrophy and distinctive cerebral demyelination. Affected individuals presented with horizontal and vertical gaze-evoked nystagmus and hyperreflexia as initial clinical signs, and a variable age of onset ranging from 12 to 60 years. Neurophysiological studies showed moderate axonal sensory polyneuropathy with altered sympathetic skin re- sponse predominantly in the lower limbs. We identified the c.1877C . T (p.Ser626Leu) pathogenic variant within the SAMD9L gene as the disease causative genetic defect with a significant log-odds score (Zmax = 3.43; θ = 0.00; P , 3.53 × 10−5). We demonstrate the mitochondrial location of human SAMD9L protein, and its decreased levels in patients’ fibroblasts in addition to mitochondrial per- turbations. Furthermore, mutant SAMD9L in zebrafish impaired mobility and vestibular/sensory functions. This study describes a novel spinocerebellar ataxia subtype caused by SAMD9L mutation, SCA49, which triggers mitochondrial alterations pointing to a role of SAMD9L in neurological motor and sensory functions. 1 Functional and Translational Neurogenetics Unit, Department of Neuroscience, Research Institute Germans Trias i Pujol (IGTP), Universitat Autònoma de Barcelona-Can Ruti Campus, Badalona, Barcelona, Spain 2 Neurology and Neurophysiology Section, Hospital Mateu Orfila, Mahón, Menorca, Spain 3 Centro Nacional de Análisis Genómico (CNAG-CRG), Center for Genomic Regulation, Barcelona Institute of Science and Technology (BIST), Barcelona, Spain 4 Neuromuscular and Functional Studies Unit, Neurology Service, University Hospital Germans Trias i Pujol (HUGTiP), Universitat Autònoma de Barcelona-Can Ruti Campus, Badalona, Barcelona, Spain 5 Neurodegenerative Diseases Unit, Neurology Service, Department of Neuroscience, University Hospital Germans Trias i Pujol (HUGTiP), Universitat Autònoma de Barcelona-Can Ruti Campus, Badalona, Barcelona, Spain 6 Parkinson Disease and Movement Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona (UB), Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED: CB06/05/0018-ISCIII), Barcelona, Spain 7 IDIBELL, L’Hospitalet, Barcelona, Spain Correspondence to: Dr Antoni Matilla-Dueñas Head of the Neurogenetics Unit Health Sciences Research Institute Germans Trias i Pujol (IGTP) Ctra. de Can Ruti, Camí de les Escoles s/n Received July 02, 2021. Revised October 20, 2021. Accepted February 08, 2022. Advance access publication February 10, 2022 © The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 | BRAIN COMMUNICATIONS 2022: Page 2 of 21 M. Corral-Juan et al. 08916 Badalona, Barcelona, Spain E-mail: amatilla@igtp.cat Keywords: spinocerebellar ataxia; SAMD9L; SCA49; mitochondria; zebrafish Abbreviations: Dpf = days post-fertilization; GEN = gaze-evoked nystagmus; IDR = intrinsically disordered region; LOD = logarithm of the odds ratio; MAPs = microtubule-associated proteins; MDS = myelodysplastic syndromes; mtDNA = mitochondrial DNA; NCV = nerve conduction velocity; SAMD9L = sterile alpha motif domain containing 9 like; SARA = scale for the assessment and rating of ataxia; SCA = spinocerebellar ataxia; SEM = standard error of the mean; SNAP = sensory nerve action potential; SNP = single nucleotide polymorphism; SSEP = somatosensory evoked potential; SSR = sympathetic skin response; TEM = transmission electron microscopy; WES = whole-exome sequencing Graphical Abstract when present, secondary signs including sensory deficits, par- Introduction kinsonism, dysphagia, cognitive dysfunction or deafness The autosomal dominant spinocerebellar ataxias (SCAs) are which often are distinctive in each specific SCA subtype.4–6 a highly heterogeneous group of rare inherited movement Variable degeneration of the cerebellum, basal ganglia, cere- disorders characterized by progressive cerebellar ataxia, of- bral cortex, optic nerve, pontomedullary systems, spinal tracts ten variably associated with pyramidal signs, parkinsonism or peripheral nerves appears to underlie the heterogeneity of or abnormal movements, lower motor neuron signs, periph- clinical signs and symptoms.4,7,8 Treatments are scarce and eral neuropathy, ophthalmoplegia, pigmentary retinopathy, mostly symptomatic.9 At least 40 loci presenting with differ- seizures or dementia.1–3 The disease onset presents typically ent pathogenic types of genetic mutations have been described in adulthood, albeit some clinical signs appear before ataxia associated with the rare Mendelian forms of SCAs, which can is clinically apparent. Over 48 well-defined SCA subtypes trigger cellular toxicity by gain-, loss-of-function effects or a have been described evidencing the high clinical and genetic combination of them, either at the protein or RNA levels heterogeneity. Common primary disease signs include cerebel- in vulnerable neurons (reviewed in Klockgether et al.2). lar ataxia, dysarthria and oculomotor abnormalities and, Identifying the genetic deficits has led to understand the New ataxia caused by a SAMD9L mutation BRAIN COMMUNICATIONS 2022: Page 3 of 21 | 3 underlying cellular and molecular pathogenic pathways impli- of them with a detailed neurological exam. The scale for the as- cated, revealing recurrent alterations of transcription, protein sessment and rating of ataxia (SARA) scores23 were assessed aggregation and clearance, autophagy, ion channel physi- for six patients (III:7, IV:2, IV:6, IV:8, IV:14 and V:1). ology, calcium homeostasis or mitochondrial defects as the Affected patients were further investigated using an enhanced main pathological mechanisms underlying the different clinical protocol, including electrocardiograms, echocardio- SCAs phenotypes.10–13 Dysregulation of mitochondrial func- grams, audiometric tests, nerve conduction studies, MRI of tion is a common pathogenic trigger of neurodegeneration, the brain and evoked potentials, during follow-up visits. also identified in spinocerebellar and spastic ataxias, ataxia syndrome, multiple system atrophy and polyneuropathy, Electrophysiological studies many of them presenting with variable degrees of ataxia, neur- opathy and hypo- or hyperreflexia.14,15 Six patients from the M-SCA family (III:7, IV:2, IV:6, IV:8, Mutations within the SAMD9L gene or its paralogue IV:14 and V:1) underwent nerve conduction, EMG and SAMD9, both genes located side-by-side on Chromosome somatosensory evoked potentials (SSEPs) studies. In three pa- 7q21, have been previously associated with a myriad of tients, we could also assess autonomic nervous system function. intra- and inter-familiar pleiotropic phenotypes.16 Germline All neurophysiological studies were performed by means of frameshift SAMD9L mutations were first described in chil- TruTrace® EMG system (DEYMED Diagnostic) and con- dren presenting with inflammation of subcutaneous fat,17 ducted according to standard methodology.24 A detailed proto- and missense gain-of-function mutations in SAMD9L in col is included in the Supplementary material. Reference control ataxia-pancytopenia syndrome (ATXPC), characterized by a values were obtained from Kimura as described.25 variable expression of a progressive neurological phenotype, pancytopenia and hypocellular bone marrow.18,19 On the Cerebellar volume quantification other hand, heterozygous missense mutations within the para- To quantify cerebellar volume, we used CERES algorithm26 logue SAMD9 have been associated with MIRAGE syndrome, to calculate the percentage of the relative volume to the total a severe early-onset condition characterized by myelodyspla- intracranial volume on T1-weighted 1.5 T MRI images from sia, infections, restricted growth, adrenal hypoplasia, genital five patients (Patients IV:2, IV:6, IV:8, IV:14 and V:1). phenotypes and enteropathy.20 The common unifying pheno- Normal boundaries were used from 30 age- and gender- type caused by SAMD9 or SAMD9L mutations is the matched controls (range: 24–75 years) randomly selected early-onset myelodysplasia syndrome with partial or complete from the open-access IXI dataset as reported.27 Monosomy 7, mainly described in paediatric cohorts without syndromic manifestation.21,22 While the penetrance of SAMD9L mutations in haematological malignancies is in- Genetic studies complete (estimated 70%),16 their pathogenic contribution Genomic DNA samples were isolated from blood leucocytes to neurological phenotypes is much lower and unclear.21 using automated DNA purification (Chemagen, PerkinElmer). In this study, we identify a SAMD9L mutation defining a DNA samples were obtained from seven affected and six un- novel SCA phenotype in kindred (M-SCA) from Menorca. affected relatives. Additionally, 80 genomic DNA samples Affected patients variably present with ataxia, nystagmus, from the Menorcan general population were analysed to dysarthria, polyneuropathy, pyramidal signs and cerebral study the SAMD9L variant frequency. Genomic mitochon- demyelination. In patients’ fibroblast and a zebrafish model, drial DNAs (mtDNAs) were sequenced using the Illumina we demonstrate the mitochondrial perturbations underlying MiSeq platform (Illumina, Inc.), and mtDNA copy number the molecular pathology in this new SCA pointing to the role was determined by qPCR. Mitochondria DNA lesions were of SAMD9L in neurological motor and sensory functions. quantified using an adapted long-run qPCR method for DNA-damage quantification.28 Detailed protocols are de- scribed in Supplementary material (Materials and Methods Patients and methods section). This study was conducted according to the ethical principles for medical research involving human subjects according to Genome-wide linkage analysis the Declaration of Helsinki. Informed consents were ob- Eleven family members, five healthy and six affected, were tained for all individuals participating in the study, which included in a genome-wide linkage study performed in col- were approved by the ethical committee of the University laboration with the Centro Nacional de Genotipado using Hospital Germans Trias i Pujol in Badalona, Spain. the Illumina Infinium HumanOmni5 Chip (Illumina, Inc.). Genotypes were assigned using the BeadStudio genotyping module software (Illumina, Inc.), and initial non-parametric Clinical studies and parametric linkage were analysed using Merlin 1.1.2.29 Clinical information from medical histories was obtained for Two-point and multipoint genetic linkage analyses were per- 37 members from the M-SCA family including 11 affected in- formed with MLINK and LINKMAP (version 5.10), from dividuals, while the clinical examination was performed in nine the Quiklink compilation (version 16) of LINKAGE.30,31 4 | BRAIN COMMUNICATIONS 2022: Page 4 of 21 M. Corral-Juan et al. We subsequently used two additional samples (V:2 and V:3) performed using HHpred.34 Protein structure was generated and informative single nucleotide polymorphisms (SNPs) with PROSITE (https://prosite.expasy.org/mydomains/).35 within the candidate region and their allele frequencies as Microtubule-binding motifs were predicted using the genetic markers. SLINK simulation program32 was MAPanalyzer36 and PredictProtein,37 DisEMBL,38 39 40 used to calculate the P-value associated with the multipoint InterPro and MobiDB-lite, were used for intrinsically logarithm of the odds ratio (LOD) score that was obtained in disordered regions (IDRs) prediction. NetPhos 3.141 and 1000 replicates of the pedigree. Both complete-penetrance NetworKIN42 servers were used to predict serine, and age-dependent penetrance models were considered threonine or tyrosine phosphorylation sites. hSAMD9L (Supplementary material, Materials and Methods section). (NP_001290425.1) reference protein was used. Protein– protein functional network interaction analysis was per- Whole-exome sequencing formed using the STRING database.43 Whole-exome sequencing (WES) was performed with genomic DNA obtained from peripheral blood of Patients IV:2 and Transmission electron microscopy IV:14 at the National Centre for Genomic Analysis (CNAG) Fibroblasts from patient IV:14 and one age- and gender-matched in Barcelona, Spain. Exome capture was performed using control were growth and fixed in 2.5% glutaraldehyde, 2% Nimblegen SeqCapEZ Exome V.3 (Roche) for 64 Mb according paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 2 h to the manufacturer’s protocol. Sequencing and bioinformatics at 4°C and processed as described.44 Ultrathin sections were analysis protocols are included in the Supplementary material. stained with uranyl acetate and lead citrate and examined in an EX-electron microscope (JEM1200; JEOL) at 80 kV. Sodium dodecyl sulphate– Pictures were acquired using from 12 to 100 K magnifications. Subcellular organelles from 20 fibroblasts sections were classi- polyacrylamide gel electrophoresis fied according to previous publications identifying autophago- and immunoblotting somes surrounded by double membrane and autolysosomes Proteins were extracted from fibroblast samples from Patients filled with undigested lipids.45,46 IV:14 and V:1 and two age-matched controls by homogeniza- tion following standard and cell fraction protocols summar- Zebrafish ataxia model ized in the Supplementary material. Primary antibodies used All applicable international, national and/or institutional were anti-β-actin (AC15; Sigma–Aldrich), anti-ATP5H guidelines for the care and use of animals were followed. (SAB4500107; Sigma–Aldrich), anti-DRP1 (sc-271583; Animal procedures were approved by the Ethics Committee Santa Cruz), anti-GAPDH (G9545; Sigma–Aldrich), on Animal Experimentation of the Germans Trias i Pujol anti-LAMP1 (H4A3; DSHB), anti-LC3 (NB100-2220; Research Institute. Zebrafish studies were conducted in col- Novus Biological), anti-MFN1 (sc-166644; Santa Cruz), laboration with Zeclinics (Supplementary material). anti-SAMD9L (25173-1-AP; Proteintech), anti-SQSTM1 Locomotion and response to visual and physical stimuli (2C11; Thermo Fisher Scientific) and anti-VDAC (D73D12; were traced and analysed by the EthoVision XT 12 software Cell Signaling). Infrared-dye conjugated secondary antibodies (Noldus) and the DanioVision (Noldus) device in 32 5-days anti-mouse IRDye-800CW and anti-Rabbit IRDye 700CW post-fertilization (dpf) zebrafish larvae from non-injected, con- (Li-Cor) were used. Signals were detected and analysed with trol injected with lynGFP, wild-type and mutant SAMD9L Odyssey analyser software (Li-Cor). groups distributed in six-well plates. To detect changes in the larvae locomotion deviations from the stereotyped behaviour Human SAMD9L expression characterized by motility in the dark phase and reduced motil- Total RNAs were obtained from fibroblast samples from ity in the light phase, larvae underwent 50 min dark/light alter- Patients IV:14 and V:1 and two age-matched controls fol- nating environments of 5 min each. This experiment was lowing standard protocols included in the Supplementary replicated with 10 min of alternating dark/light cycles. Data material. ENCODE SAMD9L RNA sequence reads were points for distances travelled (mm) during each minute and compared on BAM files from adult cerebellar Purkinje cells, the number of head turns was measured. Data outside the adult granular and pyramidal cells, 6-year-old child cerebel- 1.5 × interquartile range were considered outliers. lum and cerebellar and spinal cord human embryos.33 Statistical analysis Computational protein structure Statistical analyses were performed using SPSS 21.0 prediction and protein–protein (IBM Corp.). Statistical significance was defined as P , 0.05. The Spearman’s rank correlation coefficient for interaction analysis non-linear relationship was used to assess correlation be- Sequence alignments against NCBI Conserved Domains tween SARA scores and patients’ age. Statistical differences (CD)_v3.16, PDB_mmCIF70_4_Feb, Pfam-A_v32.0 and in fibroblast samples for ATP determination and transmis- SMART_V6.0 domain and structural databases were sion electron microscopy (TEM) studies were analysed using New ataxia caused by a SAMD9L mutation BRAIN COMMUNICATIONS 2022: Page 5 of 21 | 5 the Shapiro–Wilk test to assess for normal distribution diffuse demyelination involving periventricular and centrum followed by the parametric t-test, one-way ANOVA or alter- semiovale white matter suggestive of hypoxic leucoencepha- natively with the non-parametric Mann-Whitney U-test. For lopathy (Fig. 1E). zebrafish studies, behavioural time series statistical data were MRI of the brain showed cerebellar atrophy in three add- analysed using two-ways repeated mixed-effects ANOVA itional affected patients (IV:6, IV:8 and IV:14) with brain de- followed by post hoc comparisons with the Tukey HSD for myelination. Volumetric assessment in the five patients data groups with equal variances. Total distance travelled revealed a 28.96% significant decrease in cerebellar volume and number of turns were further analysed using one-way relative to the total intracranial volume (6.55 + 0.55%) mixed-effects ANOVA followed by post hoc comparisons compared with 30 age- and gender-matched controls with the Tukey HSD for data groups with equal variances. (Supplementary Fig. 1) with cerebellar relative volume Those values that did not meet normality or equal variances (9.22 + 0.22%; F(1,8) = 99.506; P , 0.0001; Table 2; criteria were individually tested with non-parametric test Supplementary Fig. 2), accompanied with a 36.86% de- Mann–Whitney U-test. SEM denotes for standard error of crease in cerebellar grey matter relative to the total brain the mean. grey matter (affected patients = 4.32% + 0.47; controls = 6.84% + 0.17%; F(1,8) = 122.136; P , 0.0001; Table 2; Supplementary Fig. 3). Furthermore, all affected patients Data availability presented different grades of diffuse demyelination of the The data supporting the findings of this study are available on cerebral white matter. Five patients (III:7, IV:2, IV:6, IV:8 request from the corresponding author. The data are not pub- and IV:14) presented with moderate axonal sensory licly available due to the fact that the genetic information may polyneuropathy predominantly in the lower limbs (Fig. 2A compromise the privacy of research participants. and B) compared with control values (Supplementary Fig. 4), albeit at later stages of disease progression patients also revealed reduction of sensory nerve amplitudes in upper limbs (Supplementary Fig. 4). EMG studies were Results normal in all seven patients evaluated. Tibial nerve SSEPs showed an abnormal latency of P37 response in pa- Clinical description tients diagnosed with sensory axonal polyneuropathy Eleven affected patients from the M-SCA family originally (Fig. 2C and D). In two patients (IV:2 and IV:6), sympathet- from Menorca, Spain, were clinically diagnosed by a neur- ic skin responses (SSRs) were abnormal in lower limbs ologist (P.C., N.G.R., L.I., R.A.-R.) and a detailed clinical (Fig. 2E), and one patient (IV:2) also showed an abnormal examination was performed in nine of them (Fig. 1A). R–R interval (Fig. 2G). Horizontal and vertical gaze-evoked nystagmus (GEN) was Interestingly, younger patient (V:1) presenting with hori- present as the first clinical sign in four patients (IV:8, zontal and vertical GEN, hyperreflexia and cerebellar atrophy IV:14, V:1, V:3; Table 1) in the absence of ataxia. without apparent ataxia, recorded normal electrophysiologic- Hyperreflexia was also present at the first initial clinical al values at 36 years of age. The progression of the disease is examination. Ataxia onset ranged from 30 to 60 years old slow, although variable, and the SARA scores23 in this family (Table 1). Initial nystagmus progressed with diplopia or os- follow an exponential trend (rs(4) = 0.92, P = 0.008; Fig. 1F). cillopsia in four patients (III:7, IV:6, IV:8 and IV:14; The disease progressed with dysmetria and dysarthria, and se- Table 1). Patient IV:14 also referred recurrent vertigo. verity varied among patients. Relevantly, Patients III:2 and Younger patients (V:1 and V:3) presented with horizontal III:7 became wheelchair-bound after 15 and 31 years from and vertical GEN and hyperreflexia without ataxic signs. ataxia onset, respectively. None of these patients showed fas- Patient V:1 was neurologically evaluated for the first time ciculations, epileptic seizures, cognitive impairment or haem- at the age of 12 years and disclosed horizontal and vertical atological alterations (Supplementary Fig. 5A). Partial or total GEN, hyperreflexia with bilateral Babinski’s sign, clonus haploinsufficiency on Chromosome 7 in their haematological and pes cavus. Due to family history of falls and unsteady lineages was discarded by NGS or Sanger sequencing analysis gait, clinical examination was complemented with CT which from blood white-cells DNA in their haematological lineages showed cerebellar atrophy (Fig. 1B), mega-cisterna magna (Supplementary Fig. 5B). In conclusion, for this specific SCA with discrete ventricular dilatation, suggestive of initial signs phenotype, we found GEN and hyperreflexia as characteristic- of brain demyelination (Fig. 1C). His father (IV:2) was pre- ally initial signs before ataxia onset. All five patients with neu- sented with gait instability since the age of 50. Clinical exam- roimaging data revealed diffuse cerebral demyelination ination after 15 years of disease progression revealed GEN, (Fig. 1C and E; data not shown). No extrapyramidal signs dysmetria, dysdiadochokinesia, severe dysarthria and lower were identified in any affected patient. limbs hyperreflexia with bilateral extensor plantar response. MRI at 65 years of age showed global and symmetric dila- tion of the ventricular system, lateral sulcus and cortical Genomic studies grooves with an accused diffuse vermal and hemispheric The genome-wide linkage analysis from six affected patients cerebellar atrophy (Fig. 1D). Cerebral parenchyma showed and five healthy family members from the M-SCA family 6 | BRAIN COMMUNICATIONS 2022: Page 6 of 21 M. Corral-Juan et al. Figure 1 Pedigree, MRI and SARA scale from M-SCA patients. (A) Pedigree of the five-generation family from the Balearic island of Menorca with 11 affected individuals. Black asterisks denote individuals included in genome-wide linkage studies; red asterisks denote individuals studied by WES. (B–E) Sagittal and transverse T1-weighted MRI scans of Patient V:1 brain revealing cerebellar atrophy (B), and initial signs of focal brain demyelination lesions noted by red arrows (C). Imaging of the brain of the father of patient V:1 (IV:2) also showed severe cerebellar vermis atrophy (D), and marked cerebral demyelination noted by red arrows (E) compared with age- and gender-matched controls (Supplementary Fig. 3). (F) SARA clinical scale of six affected patients showing variable severity and progression of the disease following an exponential pattern (rs(4) = 0.92, P = 0.008). New ataxia caused by a SAMD9L mutation BRAIN COMMUNICATIONS 2022: Page 7 of 21 | 7 using the Illumina Infinium HumanOmni5 Chip including Ages of onset of clinical signs, biased by the first examination, presented with ranges from 12 to 60 years. Initial clinical presentation included GEN and hyperreflexia evolving to a generally slow progressive cerebellar syndrome with unsteady gait, Wheelchair at 75 Wheelchair at 78 over 4 000 000 markers, revealed significant genetic linkage Ankle Clonus Comment Strabismus Strabismus Vertigo; to 7q21 with the highest LOD score (Zmax = 3.01; Vertigo − − − P , 0.0001) defining a 19.1 Mb candidate region flanked by the polymorphic markers rs12705836 and rs16869440 (Fig. 3A). No other significant genetic linkage regions other Dysmetria Dysarthria Diplopia Oscillopsia than SAMD9L on 7q21 were identified. WES of Patients IV:2 and IV:14 identified 445 shared heterozygous candidate − − + − − − + − − variants compatible with an autosomal dominant inherit- ance. After filtering variants by their position within the gene, population frequency, and their predicted pathogen- − − + − + + + − − icity, a missense c.1877C . T (p.Ser626Leu) variant within the fifth exon of the SAMD9L gene located on 7q21, was se- lected as the candidate pathogenic variant. This variant seg- +++ +++ +++ ++ + − − − − regated with the disease and was absent in all six healthy individuals studied from the M-SCA family, and was not identified in 80 healthy unrelated individuals from Menorca’s general population. Further linkage analysis +++ +++ ++ ++ + + + − − with two additional family members, and considering a complete-penetrance model, revealed a significant two-point LOD score between the locus trait and the identified variant Babinski proposed as the causative disease mutation (Zmax = 3.43; sign − − − − − + − + − θ = 0.00; P , 3.53 × 10−5; Table 3). The multipoint LOD score analysis assuming a complete-penetrance model with Deep tendon Hyperreflexia Hyperreflexia Hyperreflexia Hyperreflexia Hyperreflexia Hyperreflexia Hyperreflexia Hyperreflexia Hyperreflexia three SNPs (rs17393952, rs4455763 and rs10263800) in reflexes the 7q21 candidate region and the candidate variant, re- sulted in a maximum significant multipoint LOD score Zmax of 3.46 (P , 3.28 × 10−5; Supplementary material, Table 1 Clinical signs in nine affected patients from the Spanish M-SCA kindred Results section). Furthermore, linkage analysis using an age- + (V, H) + (V, H) + (V, H) + (V, H) + (V, H) + (V, H) + (V, H) + (V, H) + (V, H) GEN dependent penetrance model with five multiple liability classes also resulted in significant two-point (Zmax = 3.10; θ = 0.00; P , 7.89 × 10−5) and multipoint (Zmax = 3.12; duration Disease θ = 0.00; P , 7.51 × 10−5) LOD scores using the same mar- 7 8 1 20 36 20 14 35 28 kers. The c.1877C . T (p.Ser626Leu) (Fig. 3B) variant with- dysmetria, dysarthria, dipoplia and oscillopsia. −, absent; +, mild; ++, moderate; +++, severe. in SAMD9L gene involves a serine to leucine amino acid First clinical sign Babinski, pes cavus change, and was predicted to be deleterious by SIFT, Unsteady gait Unsteady gait Unsteady gait Unsteady gait Unsteady gait GEN, clonus, FATHMM, Mutation Assessor, Mutation Taster and GEN GEN GEN Provean algorithms. The variant is highly conserved through evolution (Fig. 3C), it is classified in the ClinVar database as a variant of uncertain significance (rs1554341671; RCV000498009.2), without minor allele frequency, and Age of ataxia identified in two related paediatric cases presenting with hy- onset pocellular marrow, dyserythropoiesis and Monosomy 7 in − − 60 58 47 50 60 60 30 the absence of ataxia or cerebellar syndrome.21 SAMD9L protein structure, domain clinical sign Age at first and protein–protein interaction 60 58 45 48 58 50 15 12 37 To investigate hSAMD9L protein structure and domains, we analysed the hSAMD9L protein sequence with HHpred pro- tein sequence profile predictor and identified protein regions Gender Age 80a 65a 81 68 66 64 50 40 38 significantly similar to SAM (Ser6-85His), AAA+ ATP-ase/ Hydrolase (711Lys–910Lys) and Apaf-1 ADP bound M M M M F F F F F (716Glu–1018Glu) domains (Fig. 3D; Supplementary Deceased. Table 4) in consonance with previously reported domain IV:14 structures in SAMD9 family members.47 Furthermore, the IV:2 IV:6 IV:8 III:2 III:5 III:7 V:1 V:3 ID analysis revealed the significant similarity with SPAST, a 8 | BRAIN COMMUNICATIONS 2022: Page 8 of 21 M. Corral-Juan et al. Table 2 MRI cerebellar volume and grey matter assessment Relative Patient Age at cerebellar volume Age- and gender-matched controls Relative grey Age- and gender-matched controls ID MRI (%) cerebellar volumes range (%) matter (%) relative cerebellar volumes range (%) IV:8 61 6.64 7.86–10.65 4.49 5.65–7.95 IV:6 59 7.05 7.93–10.72 4.64 5.70–7.99 IV:14 46 7.07 7.93–10.59 4.78 5.82–8.00 IV:2 64 5.76 7.51–10.17 3.59 5.50–7.68 V:1 36 6.26 8.10–10.75 4.12 5.99–8.16 MRI assessment in five patients revealed significant decrease in cerebellar volume and cerebellar grey matter compared with age- and gender-matched controls (Supplementary Table 3). SKD1/VPS4B, katanin and torsin-1A AAA-ATPase specific Expression and mitochondrial protein domains, implicated in microtubule and lysosomal trafficking (Supplementary Table 4). Microtubule-associated localization of human SAMD9L protein (MAP) analyser classified hSAMD9L protein as a Immunostaining with anti-SAMD9L antibody in control MAP under a 90% of specificity threshold and identified a sig- cerebellar sections revealed a punctate staining mainly in nificantly enriched motif in MAPs proteins, S….LKS between Purkinje cell soma in the human cerebellar cortex and multi- amino acids 619–626 (STILKLKS) where the identified muta- polar neurons of the cerebellar dentate nucleus (Fig. 4A), tion c.1877C . T entails the p.Ser626Leu amino acid change similar to mitochondrial ATP5B protein staining (Fig. 3E; Supplementary Table 5). (Supplementary Fig. 9), indicative of mitochondrial localiza- Comprehensive protein analysis with structural predictors tion. Antibody specificity control staining is included in identified an intrinsic disorder (ID) region around the Supplementary Fig. 9. Some basket cells also appeared SAMD9L amino acid residue Ser626 (Supplementary Fig. 6). slightly stained. SAMD9L staining was also examined in The SAMD9L amino acid change p.Ser626Leu alters the fibroblast cells from age-matched control (Fig. 4B) and ID region predicted by PredictProtein (PROFBval and one affected patient (Fig. 4C; Supplementary Fig. 10), re- Ucon) and DisEMBL (hot loops) algorithms, and it vealing mitochondrial staining and co-localization to the abolishes the identified ID region according to the predic- mitochondrial marker mitoTracker Red CMXRos. No sig- tion by InterPro (MobiDB-lite) (Supplementary Table 6). nificant differences in the mitochondrial network were ob- Furthermore, PredictProtein (PROFAcc) identified the served in patients’ fibroblasts compared with controls 624–629 protein region of SAMD9L as a solvent-exposed (Supplementary material, Results section). ENCODE region (Supplementary Fig. 6) and NetPhos 3.1 and RNAseq analysis confirmed relative expression of NetworKIN algorithms predicted the residue Ser626 with- SAMD9L in adult Purkinje cells and almost no expression in SAMD9L to be potentially phosphorylated by protein in cerebellar granule and pyramidal cells (Supplementary kinase C (Supplementary Fig. 7), a kinase directly impli- Fig. 11). RNA tissue expression also showed higher levels cated in cerebellar neurodegeneration in the SCA 14 of SAMD9L in child cerebellum and in embryonic cerebel- subtype.48 lum and spinal cord, in consonance with stages of high With the STRING algorithm, we studied the interactions mitochondrial activity and tissues affected in the pathogen- among SAMD9L and KATNA1, SPAST, VPS4B proteins esis of this new ataxia subtype. identified by the HHpred protein sequence profile predictor Immunoblotting of lysed and cellular fractioned fibro- algorithm (Supplementary Table 4), and among the previ- blasts confirmed SAMD9L mitochondrial localization and ously reported Samd9l-interacting proteins in mice Eea1 showed decreased SAMD9L protein levels (Fig. 5A; and Rab549 in the context of the whole human genome Supplementary Figs 18 and 19). To corroborate these find- (Supplementary Fig. 8). The SAMD9L and SPAST network ings, total cellular extraction from lysed fibroblasts was im- interaction was identified via IFI44I, also known as MAP munoblotted and reduced levels of SAMD9L in fibroblasts 44, and ATL1 proteins. The protein–protein interaction en- from affected patients were found (Fig. 5B; Supplementary richment analysis showed significantly more interactions Fig. 20) without alteration of SAMD9L cDNA levels among these proteins than expected (P , 2.52 × 10−7). (Fig. 5C; Supplementary Fig. 21). Co-expression and interaction analyses linked both IFI44I and EEA1 with SAMD9L and revealed an association of the SAMD9L protein network with MAPs and endosomal/ Mitochondrial perturbations in lysosomal pathways. Interestingly, mutations in the ATL1, SPAST, RAB7A or KATNB1 genes have been previously as- patients’ fibroblasts sociated with diseases presenting with hyperreflexia, sensory Potential alterations of fibroblasts’ mtDNAs were analysed axonal neuropathy or sensory impairment among other for mtDNA depletions and found an increased copy number signs, which overlap with clinical signs identified in this of the D-LOOP mitochondrial genomic region compared new ataxia subtype in our family (Supplementary Table 7). with the MT-CO3 region, suggesting increased activity of New ataxia caused by a SAMD9L mutation BRAIN COMMUNICATIONS 2022: Page 9 of 21 | 9 Figure 2 Moderate axonal sensory polyneuropathy predominantly in the lower limbs and abnormal SSR and R–R interval in affected patients III:7, IV:6 and IV:2, respectively. (A and B) Moderate axonal sensory polyneuropathy predominantly in the lower limbs in IV:2. At later stages of disease progression, patients revealed reduction of sensory nerve amplitudes also in upper limbs (Supplementary Fig. 4). Tibial nerve SSEPs showed an abnormal latency of P37 response in IV:2 (C) and III:7 (D). (E) Abnormal SSR in lower limbs displayed abnormality in two patients (IV:2 and IV:6). (G) Altered R–R interval in Patient IV:2. 10 | BRAIN COMMUNICATIONS 2022: Page 10 of 21 M. Corral-Juan et al. Figure 3 Genome-wide linkage analysis in combination with whole-exome NGS sequencing identifies the c.1877C . T (p.Ser626Leu) mutation within the SAMD9L gene on 7q21 as the causative mutation. (A) LOD score plots for chromosomal region 7q21. (B) The c.1877C . T (p.Ser626Leu) SAMD9L variant is predicted as deleterious by six in silico algorithms. (C) The mutated amino acid was found highly conserved. (D) HHpred protein sequence profile predictor identified protein motifs significantly similar to SAM, AAA+ ATP-ase/ Hydrolase and Apaf-1 ADP bound domains. SAMD9L contains motifs enriched in microtubule-binding proteins. (E) The p.Ser626Leu localizes within the microtubule predicted motif S….LKS in human SAMD9L. mitochondrial replication indicative of mitochondrial bio- mitochondrial alterations suggestive of mitochondrial stress, genesis (Fig. 5D). In affected fibroblast cell lines, the ATP no significant DNA alterations were identified in the mito- concentration was found increased compared with those chondrial genomes from fibroblasts’ patients, discarding fur- from age-matched control fibroblasts (Fig. 5E). Despite these ther mtDNA damage (Supplementary Fig. 12).50 New ataxia caused by a SAMD9L mutation BRAIN COMMUNICATIONS 2022: Page 11 of 21 | 11 Table 3 Two-point LOD score Two-point LOD score θ 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.5 Z (θ) 3.43 3.13 2.819 2.48 2.13 1.76 1.37 0.97 0.57 0 The maximum LODs score was obtained with the c.1877C . T (p.Ser626Leu) variation within the SAMD9L gene on 7q21 (Zmax = 3.43, θ = 0.00; P , 3.53 × 10−5). To further investigate mitochondrial perturbations, we found that it was increased in patients’ fibroblasts despite immunoblotted with anti-ATP5H, a critical component of normal levels of VDAC protein located in the mitochondrial the energy-producing apparatus in eukaryotic cells,51 and outer membrane (Fig. 6A and B; Supplementary Figs 22–27). Figure 4 Mitochondrial localization of SAMD9L in human cerebellar Purkinje cells and multipolar neurons of the dentate nucleus and fibroblasts. (A) Immunostaining with anti-SAMD9L antibody in human control cerebellar sections revealed a punctate staining mainly in Purkinje cell soma (left) and in multipolar neurons (right) of the human dentate nucleus indicative of mitochondrial staining. Black arrows point to SAMD9L staining in Purkinje and multipolar neurons. (B and C) Immunofluorescence staining of fibroblasts from human control (B) and a M-SCA affected patient (C) showed co-localization of SAMD9L to mitoTracker Red CMXRos demonstrating mitochondrial co-localization. Magnification bars: 50 µm (A), 20 µm (B and C). 12 | BRAIN COMMUNICATIONS 2022: Page 12 of 21 M. Corral-Juan et al. Figure 5 Reduced SAMD9L protein levels and mitochondrial alterations in M-SCA patients’ fibroblasts. (A) Immunoblotting of lysed and subcellular fractioned fibroblasts confirmed SAMD9L mitochondrial localization, and decreased SAMD9L protein levels in the mitochondrial (A) and total (B) cellular extracts from patients’ fibroblasts samples (N = 2) without alteration of SAMD9L cDNA levels compared by qRT-PCR (C) [N = 2 fibroblasts samples for each group of individuals (patients and controls); three technical replicates each for SAMD9L and three technical replicates each for GAPDH housekeeping gene expression]. (D) Increased copy number of the mtDNA D-LOOP mitochondrial genome region in patient’s fibroblasts samples is suggestive of an increased activity of mtDNA replication. (E) A trend of increased ATP was found in mitochondrial fractions of affected fibroblasts [N = 2 fibroblasts samples for each group of individuals (patients and controls); four technical replicates each] compared with age-matched controls [N = 2 fibroblasts samples for each group of individuals (patients and controls); four technical replicates each]. Each point represents relative ATP levels compared with controls after normalizing for protein concentration. Controls values were set to 1 (dotted line). New ataxia caused by a SAMD9L mutation BRAIN COMMUNICATIONS 2022: Page 13 of 21 | 13 Because ATP5H plays a crucial role in regulating apoptosis, Additionally, the number of head turns during the five this evidence further supports the upregulation of ATP syn- light–dark cycles was significantly increased in thase identified. Additionally, DRP1 protein levels also ap- WT-SAMD9L compared with the control group (Mann– peared to increase. Remarkably, DRP1 is a dynamin-like Whitney U = 216, P = 0.010; Fig. 7B), and significantly de- GTPase involved in the mitochondrial division during mito- creased in S626L-SAMD9L zebrafish larvae compared with chondrial biogenesis, which has also been associated with the WT-SAMD9L (Mann–Whitney U = 192.5, P = 0.022; lysosomes in brain52 and implicated in Purkinje cells mito- Fig. 7B) demonstrating vestibular and sensory impairment chondrial transport.53 Related to lysosomes and autophagy, by the mutation in SAMD9L. This evidence of higher func- the lysosomal-associated membrane protein LAMP1, and tional activity of the human wild-type SAMD9L protein the autophagy-related proteins LC3-II and p62/SQSTM1 was not caused by RNA overexpression (Fig. 7C). also appeared overexpressed in patients’ fibroblasts Immunoblotting of lysed cellular fractions corroborated (Fig. 6A and B). Overall, these data demonstrate SAMD9L recombinant human SAMD9L mitochondrial localization mitochondrial localization, suggest an increase of mtDNA in zebrafish larvae (Fig. 7D; Supplementary Fig. 16). replication and biogenesis, and point to dysregulation of Furthermore, the levels of the mitochondrial fusion protein the lysosomal/autophagy pathway in this novel SCA subtype DRP1 were found increased in mutant SAMD9L compared caused by the SAMD9L mutation. with either wild-type or controls in zebrafish embryos To investigate subcellular structural changes, ultrastruc- (Fig. 7D). Increased DRP1 is suggestive of mitochondrial ture images from fibroblasts obtained from Patient IV:14 ob- biogenesis. tained by TEM, showed diffused mitochondrial crests Whole-mount immunofluorescence of zebrafish larvae (Fig. 6C), evidence of mitophagy (Fig. 6D) and dilated endo- showed SAMD9L mitochondrial staining in the zebrafish plasmic reticulum (Fig. 6E and F; Supplementary Fig. 13). spinal cord and peripheral nerves (Supplementary Fig. 17) Significant higher number of lysosomes in affected fibro- and in the hindbrain (Fig. 7E), co-localizing with the blasts was also identified [F(1,18) = 6.135, P = 0.023; ATP5B mitochondrial marker (Fig. 7F) and parvalbumin Fig. 6G; Supplementary Fig. 13]. No differences in the num- (Fig. 7E) in WT-SAMD9L and S626L-SAMD9L zebrafish ber of mitochondria were observed. larvae neurons. Parvalbumin showed positive immunostain- ing in inhibitory and excitatory interneurons and motoneur- ons in the zebrafish spinal cord.55 The c.1877C . T (p.Ser626Leu) mutation in SAMD9L impairs locomotion and neurosensorial Discussion phenotypes in zebrafish In the present study, we describe a new SCA subtype in nine Four putative genomic regions containing the candidate affected individuals of a five-generation family of Menorcan human SAMD9L ortholog gene sequence were identified in ancestry, characterized by ataxia, horizontal and vertical zebrafish by using meta-analysis data from previously gener- GEN, dysarthria, polyneuropathy, pyramidal signs and cere- ated transgenic zebrafish models (http://zfin.org), and com- bral demyelination as distinctive features. We identified the pared with zebrafish cerebellar RNA-seq data from the c.1877C . T (p.Ser626Leu) mutation within the fifth exon SRA database54 (access: SRX4184229; Supplementary of the gene encoding for the sterile alpha motif domain con- Fig. 14). All four regions presented low predicted translated taining 9 like protein SAMD9L as the genetic cause under- protein identity (,30%) with that of human SAMD9L and lying this new SCA. The functional studies in patients’ did not show conservation for the Ser626 amino acid. For fibroblasts demonstrated for the first time the mitochondrial this reason, transient expression of the human wild-type localization of human SAMD9L protein and mitochondrial and mutated SAMD9L mRNAs was generated in zebrafish perturbations underlying the molecular pathology in this instead of knocking out or down the endogenous zebrafish new ataxia subtype. ortholog candidates. The clinical phenotype of this new SCA subtype is charac- Accumulated survival at 24 and 96 h post-fertilization terized by early horizontal and vertical GEN and hyperre- was not significantly impaired with any mRNA concentra- flexia, which precede the sensory polyneuropathy and tion tested, and the teratogenic rate did not show any con- ataxia symptoms. Whereas GEN in all directions indicates centration dependency (Supplementary Fig. 15). Mean cerebellar dysfunction having multiple causes in cerebellar distance travelled was significantly reduced in five dpf ataxias, purely vertical GEN is caused by midbrain lesions fish exposed to SAMD9L-S626L (N = 24) compared with and purely horizontal GEN is due to pontomedullary affect- the wild-type SAMD9L group (N = 26) under dark ation.56 Albeit GEN is a common feature sign in many SCAs intervals of high locomotor activity [F(1,48) = 4.745, P = subtypes, including SCA1,57,58 SCA3/MJD,59 SCA5,60 0.034; Fig. 7A]. Furthermore, fish overexpressing wild-type SCA6,61 SCA10,62 SCA11,63 SCA13,64 SCA14,65 SCA15/ SAMD9L showed a significantly increase in mean distance 16,66 SCA18,67 SCA19/22,68 SCA20,69 rarely in SCA23,70 moved in the dark compared with control injected group SCA25,71 SCA27,72 SCA28,73 SCA31,74 frequently in (N = 28) [F(1,52) = 6.583, P = 0.013; Fig. 7A]. SCA36 (in 30% of the cases),75 SCA3876 and SCA42 (only 14 | BRAIN COMMUNICATIONS 2022: Page 14 of 21 M. Corral-Juan et al. Figure 6 Mitochondrial and lysosome/autophagy alterations in fibroblasts from ataxia patients. (A and B) The ATP5H and DRP1 mitochondrial associated proteins levels were found increased without changes in VDAC and MFN1 suggestive of mitochondrial biogenesis. Furthermore, lysosomal LAMP1 together with SQSTM1 and LC3-II protein levels were found increased suggestive of mitophagy. LAMP1, MFN1, SQSTM1, ATP5H AND LC3 proteins levels were normalized to actin B (ACTIN) and DRP1 proteins levels to GAPDH (Supplementary Figs 22–27). Controls values were set to 1 (dotted line). TEM showed diffused crests in the mitochondrial matrix (C) together with some mitophagy (D) and dilated endoplasmic reticulum (E and F) compared with age-matched control (Supplementary Fig. 13). (G) One-way ANOVA confirmed significantly increased number of lysosomes on patients’ fibroblasts samples (N = 10) compared with controls [N = 10; F(1,18) = 6.135, P = 0.023] (Supplementary Fig. 13). Black arrowheads point to mitochondria; white arrowheads point to the dilated endoplasmic reticulum. Asterisks indicate autophagosomes. Magnification bars: 0.5 µm (C, D and F), 1 µm (E). Asterisk denotes significance at P , 0.05. Vertical bars denote SEM. New ataxia caused by a SAMD9L mutation BRAIN COMMUNICATIONS 2022: Page 15 of 21 | 15 Figure 7 Mutant Ser626Leu SAMD9L triggers locomotive and neurosensory impairment and co-localizes with ATP5B or parvalbumin in zebrafish neurons. (A) One-way ANOVA with the mean distance travelled (mm) during five dark periods of high activity revealed significantly increase in the WT-SAMD9L zebrafish (N = 26) compared with the lynGFP control group (N = 28) [F(1,52) = 6.583, (continued) 16 | BRAIN COMMUNICATIONS 2022: Page 16 of 21 M. Corral-Juan et al. horizontal),77 early GEN has been only described in SCA3/ Multiple demyelinating lesions in the periventricular subcor- MJD78 and SCA6.79 Hyperreflexia was present in all affected tical white matter have only been described in SCA9 patients patients of the M-SCA family. Hyperreflexia is a common from a large multigenerational American family.105 sign in the SCAs and is present in SCA1,80 SCA2 (16%),81 Consequently, SCA1, SCA3, SCA6, SCA11, SCA14, SCA3,82 SCA6 (50%),61 SCA7,83 SCA8,84 SCA10 SCA15/16 and SCA28 should all be considered in the differ- (7,5%),82 SCA11,85 SCA12,86 occasionally in SCA13,87 ential clinical diagnosis, but when MRI data also reveal early SCA14,88 SCA15/16,89 SCA17,90 SCA19/22 (in 3/8 pa- brain demyelinating lesions, SAMD9L mutations should be tients),91 rarely in SCA23 (in 4/13 patients),92 SCA28,73 first tested. Besides brain demyelinating lesions, GEN and SCA30,93 SCA34,94 SCA35,95 SCA36,96 SCA4097 and hyperreflexia also appeared early at the pre-symptomatic SCA42.77 In contrast, bilateral Babinski sign was identified stages preceding ataxia symptoms in this novel SCA subtype. in two patients indicative of pyramidal lesion. Herein, we provide evidence that the missense mutation Abnormal sensory nerve conduction has been previously c.1877C . T (p.Ser626Leu) within the fifth exon of the described in several SCA subtypes. In SCA1, there is reduced SAMD9L gene decreases SAMD9L protein levels along or absent sensory nerve action potential (SNAP) amplitude with increased ATP5H, DRP1 and ATP evidencing mito- with or without reduced nerve conduction velocities chondrial dysregulation in patients’ fibroblasts. (NCVs); in SCA2, reduced or absent SNAP amplitude with Furthermore, we describe for the first time the mitochondrial reduced NCVs; in SCA4 and SCA25, absent SNAPs; and in localization of human SAMD9L protein in human fibro- SCA3, SCA8, SCA12 and SCA17, reduced SNAP amplitudes blasts and zebrafish. with normal NCVs.98 SSR along with R–R interval altera- To date, 35 different mutations have been identified with- tions have been previously reported only in SCA299,100 and in SAMD9L gene on Chromosome 7q21 in 93 patients from SCA3,101 where they correlate closely with the disease func- 44 unrelated families variably associated with haematologic- tional stage. This seems to occur in our M-SCA patients al malignancies including cytopenias or myelodysplastic syn- where only older patients presented with altered SSRs and drome (MDS),21,22,106–109 ATXPC with neurological R–R interval variation indicative of autonomic dysfunction. manifestations18,19,110–114 or infantile autoinflammatory It is important to note that none of our patients presented disease.17 All those patients with SAMD9L-related haemato- with comorbidities that could interfere with the result of logical malignancies presented with, in addition to the autonomic nervous system tests as diabetes mellitus. SAMD9L mutation, either uniparental monosomy of Interestingly, patients with mutations in the SPAST gene as- Chromosome 7, partial haploinsufficiency del(7q) including sociated with spastic paraplegia type 4 (SPG4) also present SAMD9L, or somatic driver mutations within SETBP1, with SSR alterations and sudomotor dysfunction.102 ASXL1, RUNX1, PTPN11, KRAS, CBL, EZH2, ETV6, Relevantly, like our patients, some SPG4 patients also BRAF or RAD21 genes.22 It seems plausible that for devel- show hyperreflexia and pyramidal signs with cerebellar atax- oping MDS, SAMD9L mutations need to occur along with ia.103 Some mutations in spastin protein causing SPG4 have Monosomy 7, either partial or total del(7q) haploinsuffi- been associated with microtubules and lysosomal traffick- ciency, or with mutations in those driver genes. In consonance, ing,104 and it contains an AAA-ATPase domain predicted our SAMD9L ataxia patients did not reveal abnormal blood to be significant similar with the AAA-ATPase domain in cell counts, MDS or cytopenia in their longitudinal medical re- SAMD9L identified in this study. This evidence points to cords, or loss of c.1877C . T SAMD9L heterozygosity de- common cellular pathways and pathophysiological disease tected by NGS or Sanger sequencing from blood white-cells mechanisms. DNA, thus ruling out SAMD9L partial or total haploinsuffi- A distinctive feature of this new SCA subtype is the pres- ciency in their haematological lineages. This may explain the ence of early diffuse cerebral demyelination which is a neu- absence of haematological malignancy in our patients. Thus, roimaging feature scarcely observed in other SCAs. ours is the first family reported to date with a SCA phenotype Figure 7 Continued P = 0.013], and significantly decreased in S626L-SAMD9L zebrafish (N = 24) compared with the WT-SAMD9L group [F(1,48) = 4.745, P = 0.034]. (B) The number of head turns during five light–dark cycles of high activity was significantly increased in WT-SAMD9L compared with the control group (Mann–Whitney U = 216, P = 0.010), and significantly decreased in S626L–SAMD9L zebrafish larvae compared with the WT-SAMD9L (Mann–Whitney U = 192.5, P = 0.022), indicative of vestibular and sensory impairment in mutant animals. (C) SAMD9L cDNA expression did not show differences between SAMD9L-WT and SAMD9L-S626L groups compared by qRT-PCR, meaning a pool of five embryos for each group; three technical replicates (three values/group) each for SAMD9L normalized to zebrafish tbp housekeeping gene expression. The average of the three technical replicates was considered as single data point for the statistical analysis (Supplementary Fig. 28). (D) Immunoblotting of lysed cellular fractions corroborated overexpression of either wild-type or mutant SAMD9L and the mitochondrial localization in zebrafish. DRP1 protein levels were found increased in mutants SAMD9L (N = 5) compared with either wild-type (N = 5) or control (N = 5) (Supplementary Figs 29–31). Whole-mount zebrafish immunofluorescence of WT-SAMD9L and S626L-SAMD9L showed mitochondrial staining in the zebrafish spinal cord and peripheral nerves (Supplementary Fig. 16) and in the hindbrain (E), co-localizing with the ATP5B mitochondrial marker (F) and parvalbumin (E) in WT-SAMD9L and S626L-SAMD9L zebrafish larvae neurons. Magnification bars: 20 µm. Asterisk denotes significance at P , 0.05. Vertical bars denote SEM. New ataxia caused by a SAMD9L mutation BRAIN COMMUNICATIONS 2022: Page 17 of 21 | 17 caused by a SAMD9L mutation without the presence of any vacuoles. This evidence would support the mutation in pre- haematological pathology. This supports the hypothesis pro- venting proper protein folding leading to abnormal mito- posed that primary SAMD9L mutations do not lead per se chondria clearance by mitophagy and compensatory to tumour cell proliferation, but that a secondary genomic mitochondrial biogenesis. The suggestive increased levels event consisting of either Chromosome 7 monosomy or som- of p62/SQSTM1, DRP1, LAMP1 and LC3-II together with atic mutations in driver genes would be needed to predispose the increased copy number of the mtDNA D-LOOP mito- to MDS and haematological malignancy.19,107 chondrial genomic region in patient’s fibroblasts support The p.Ser626Leu mutation identified in SAMD9L in our this hypothesis. patients localizes to a predicted protein IDR. Over 20% of SAMD9L shows sequence similarity to VPS4B, a protein human disease mutations occur in IDRs,115 and Serine resi- implicated in lysosomal/endosomal membrane trafficking, due is the third most common amino acid present in regulating the maturation of autophagosomes to degradative IDRs.116 Furthermore, IDRs are strongly associated with autolysosomes, a process also implicating Rab7.126,127 Rab7 mitochondrial localization and function.117 IDRs are poly- belongs to Rab GTPases proteins which are key regulators of peptide segments typically containing a higher proportion multivesicular body maturation from early endosomes, as of polar or charged amino acids and do not contain sufficient well as the fusion of multivesicular bodies mediated by lyso- hydrophobic amino acids to mediate co-operative fold- somal degradation.128,129 Another Rab GTPase, Rab5, med- ing.118 Mutations within IDR domains, such as those within iates the signalling for endosome internalization entailing the IDR of the ITPR1 protein associated with SCA15/29, ap- neurite outgrowth and dendritic branching,130 regulates en- pear to trigger pathogenicity by creating dileucine motifs af- dosomes motility by stimulating their stable association with fecting protein interactions.119 Our IDR c.1877C . T microtubules,131 co-localizing with SAMD9L in mouse lung SAMD9L mutation promotes a serine (hydrophobic and po- fibroblasts and KL cells.49 Rab GTPases and associated lar) to leucine (hydrophilic and non-polar) amino acid membrane trafficking are implicated in different neurode- change at the 626 aa position. This may trigger a similar ef- generative diseases as Charcot–Marie–Tooth, amyotrophic fect like that in SCA15/29. lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s dis- Several lines of evidence support mitochondrial perturba- ease and Huntington’s disease,129 and dysregulation of the tions triggered by the SAMD9L mutation in our ataxia pa- lysosomal pathway in the pathogenesis of SCAs including tients. We demonstrate for the first time the subcellular Machado-Joseph disease,132 SCA6,133 SCA7134 and localization of human SAMD9L protein to mitochondria SCA21.135 To date, only mutations in the AFG3L2 gene cod- in zebrafish and human cerebellar Purkinje cells and fibro- ing for mitochondrial protein ATP-dependent metallopro- blast cell lines. Likewise, the analysis of human ENCODE tease are associated with a dominantly inherited ataxia, the RNA data corroborated the expression of SAMD9L pre- SCA subtype 28 (SCA28),136 albeit mitochondrial dysfunc- dominantly in Purkinje cells, cerebellum and spinal cord, tion is a common pathogenic mechanism underlying at least which are the main tissues affected in this new ataxia sub- SCA1, SCA2, SCA3, SCA7, SCA10 and SCA12.137–142 type. To investigate how the c.1877C . T (p.Ser626Leu) To demonstrate in vivo pathogenicity of the c.1877C . T SAMD9L mutation triggers mitochondrial dysregulation, (p.Ser626Leu) SAMD9L mutation, we evaluated the mo- we used in silico algorithm which showed that the 626Ser lecular implication of SAMD9L and the mutation on loco- in SAMD9L protein locates in the predicted S….LKS motion and neurosensory functions in zebrafish. Similar microtubule-binding motif, which is highly conserved along effects on zebrafish phenotype by overexpressing wild-type primate species and is absent in the hSAMD9 protein para- proteins, such as in our study, were previously described log. Remarkably, homology and structural predictor algo- for other genes involved in neurodegenerative diseases such rithms predicted an AAA-ATPase domain within SAMD9L as SPG and neuropathy, ATL1 gene; ALS, SOD1 and protein similar to those in spastin, VPS4B (also known as TDP43 genes143–145; as well as with genes associated with SKD1), katanin and torsin-1A, which are all associated ataxia including RNF170 and VLDLR.146,147 Based on with microtubules or have been involved in lysosomal traf- our evidence, we propose a dominant negative or haploinsuf- ficking.120–122 Mutations within the SPAST gene have been ficiency effect triggered by the mutation within SAMD9L– previously associated with autosomal dominant SPG4123 S626L in zebrafish when compared with the wild-type protein presenting with hyperreflexia and pyramidal signs, with when both are similarly overexpressed for comparison pur- some patients also manifesting cerebellar ataxia.103 poses. In fact, autosomal dominant mutations in residues lo- Furthermore, some spastin mutations including R115C, cated in molecular interfaces, such as our mutation within N184T, L195V and N386K have been identified disrupting SAMD9L, predominantly trigger negative effects by affecting lysosomal function,104 and to alter interaction with microtu- protein–protein interactions.148 We demonstrate that the bules entailing to the abnormal cellular distribution of mito- SAMD9L mRNA mutation in zebrafish impaired mobility chondria, suggesting organelle transport alteration on the and vestibular/sensory functions compared with the wild-type, microtubule cytoskeleton, including transport to distal ax- pointing to the role of SAMD9L in neurological motor and sen- ons.124,125 Ultrastructural alterations were identified in the sory functions, which were both found altered in our patients. patient’s fibroblasts including markedly dilated RER, diffuse In conclusion, in this study, we describe a novel SCA sub- mitochondrial crests and the presence of mitophagy type, SCA49, caused by a c.1877C . T (p.Ser626Leu) 18 | BRAIN COMMUNICATIONS 2022: Page 18 of 21 M. Corral-Juan et al. mutation within the gene encoding for the sterile alpha motif 4. Parodi L, Coarelli G, Stevanin G, Brice A, Durr A. Hereditary atax- domain containing 9 like protein SAMD9L. Based on the ias and paraparesias: Clinical and genetic update. Curr Opin Neurol. 2018;31(4):462–471. evidence provided, we propose SAMD9L as a protein in- 5. Jacobi H, du Montcel ST, Bauer P, et al. Long-term disease pro- volved in mitochondrial function underlying neurological gression in spinocerebellar ataxia types 1, 2, 3, and 6: A longitu- motor and sensory functions in this new SCA subtype. dinal cohort study. Lancet Neurol. 2015;14(11):1101–1108. 6. de Silva R, Greenfield J, Cook A, et al. Guidelines on the diagnosis and management of the progressive ataxias. Orphanet J Rare Dis. 2019;14(1):51. Acknowledgements 7. Meira AT, Arruda WO, Ono SE, et al. Neuroradiological findings in the spinocerebellar ataxias. Tremor Other Hyperkinet Mov. We thank all patients and family members participating in this 2019;9:1–8. study. We acknowledge the IGTP-HUGTP biobank integrated 8. Rüb U, Schöls L, Paulson H, et al. Clinical features, neurogenetics in the Spanish National Biobanks Network of the Instituto de and neuropathology of the polyglutamine spinocerebellar ataxias Salud Carlos III (PT13/0010/0009), and the IGTP microscopy type 1, 2, 3, 6 and 7. Prog Neurobiol. 2013;104:38–66. 9. Matilla-Dueñas A, Infante J, Serrano-Munuera C, and genomics core facilities and staff for their contribution Ivánovic-Barbeito Y, Alvarez R, Sánchez I. Novel therapeutic chal- to this publication. Next-generation sequencing and ana- lenges in cerebellar diseases. In: Manto M Schmahmann JD Rossi F lysis were performed at the Centro Nacional de Análisis Gruol DL, Koibuchi N, eds. Handbook of the cerebellum and cere- Genómico (CNAG-CRG) and funded by the CNAG’s 2013 bellar disorders. Springer International Publishing; 2020:1–33. call ‘300 exomes to elucidate rare diseases’. We are grateful 10. Huang M, Verbeek DS. Why do so many genetic insults lead to Purkinje Cell degeneration and spinocerebellar ataxia? Neurosci to Josep Manel Rebled, Eva Prats of the Universitat de Lett. 2019;688:49–57. Barcelona electron microscopy unit for assistance with TEM 11. Paulson HL, Shakkottai VG, Clark HB, Orr HT. Polyglutamine and Dr Carles Enrich for helpful discussions on TEM images. spinocerebellar ataxias—from genes to potential treatments. Nat SNP genotyping was provided by the Human Genotyping Unit Rev Neurosci. 2017;18(10):613–626. at the USC-Universidad de Santiago de Compostela of the 12. Jones L, Houlden H, Tabrizi SJ. DNA repair in the trinucleotide re- peat disorders. Lancet Neurol. 2017;16(1):88–96. Centro Nacional de Genotipado (CEGEN). We also thank 13. Matilla-Dueñas A, Ashizawa T, Brice A, et al. 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