Assessment of a Targeted Gene Panel for Identification of Genes Associated with Movement Disorders

Supplementary Online Content

Montaut S, Tranchant C, Drouot N, et al; French Parkinson’s and Movement Disorders Consortium. Assessment of a targeted gene panel for identification of genes associated with movement disorders. JAMA Neurol. Published online June 18, 2018. doi:10.1001/jamaneurol.2018.1478

eMethods. Supplemental methods.

eTable 1. Name, phenotype and inheritance of the genes included in the panel.

eTable 2. Probable pathogenic variants identified in a cohort of 23 patients with cerebellar ataxia using WES analysis.

eTable 3. Negative cases in a cohort of 23 patients with cerebellar ataxia studied using WES analysis.

eTable 4. Variants of unknown significance (VUSs) identified in the cohort.

eFigure 1. Examples of pedigrees of cases with identified causative variants.

eFigure 2. Pedigrees suggesting mendelian inheritance in negative cases.

eFigure 3. Examples of pedigrees of cases with identified VUSs.

eResults. Supplemental results.

This supplementary material has been provided by the authors to give readers additional information about their work.

Downloaded From: https://jamanetwork.com/ on 10/02/2021 eMethods. Supplemental methods

Patients selection

In the multicentric, prospective study, patients were selected from 25 French, 1 Luxembourg

and 1 Algerian tertiary MDs centers between September 2014 and July 2016. Inclusion

criteria were patients (1) who had developed one or several chronic MDs (2) with an age of

onset below 40 years and/or presence of a family history of MDs. Patients suffering from

essential tremor, tic or Gilles de la Tourette syndrome, pure cerebellar ataxia or with

clinical/paraclinical findings suggestive of an acquired cause were excluded. Most of the

patients underwent common biochemical testing (copper, ceruloplasmin), brain imaging and

most common relevant genetic testing (such as Wilson’s disease or Huntington’s disease)

before their inclusion. Demographic, clinical and paraclinical data were collected, as well as

family history and a family tree. For all patients, a written informed consent for genetic

testing was obtained, either from adult probands or from the legal representative in case of

minors and the study was approved by the local ethics committee.

Targeted genes and capture design

An exhaustive literature review was performed in order to design a targeted genes panel made

up of 127 genes known to be either candidates, or clearly involved in MDs (Supplementary

table 1). Some genes included were considered as candidate genes since the pathogenicity of

their mutations was not clear 6, since they were suspected to act as risk factors 2, or because

they are classically responsible for a different phenotype than MDs 7. The total size of

targeted regions, all exons, 50-bp flanking sequences of target exons (RefSeq database, hg19

assembly) encompassed 753 kb (kilobases).

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Library preparation, targeted capture and sequencing

Genomic DNA samples were extracted from peripheral blood samples, prepared and

controlled following standard procedures in the Genetics and Cellular and Molecular Biology

Institute (IGBMC, Illkirch, France). The capture design was performed with SeqCap EZ

Designs following the manufacturer’s instructions (Roche, Madison, WI). DNAs (1µg) were

sheared mechanically using Covaris E210 (duty cycle: 10%; intensity: 5; cycles per burst:

200; time: 340s) (Woburn, MA). Sequencing and multiplexing adaptors were added

simultaneously on 900 ng of sheared DNA using the KAPA Library Preparation Kit (KAPA

Biosystems, Wilmington, MA) and the NimbleGen™ SeqCap Adapter Kits A and B (Roche,

Madison, WI). After amplification and quality assessments, in-solution targeted capture and

post-hybridization amplification were performed on 1µg of multiplexed DNA-prepped

libraries using the NimbleGen™ SeqCap EZ Accessory Kit v2 and Pure Capture Bead Kit.

Steps of washing, purification and elution were performed using Agencourt SPRI® AMPure

XP (Beckman Coulter, Brea, CA). Finally, paired-end sequencing was performed on a

NextSeq550 platform (Illumina, Inc., San Diego, CA), multiplexing up to 24 samples per

sequencing lane.

Bioinformatic pipeline

Read mapping and variant calling were performed following standard procedures. Variant

filtering was performed using Polyweb, a computer interface developed by the Université

Paris Descartes which collects variant-specific information to rank them according to their

predicted pathogenicity using data bases (Exome Variant Server (EVS), Exome Aggregation

Consortium (ExAC), Single Nucleotide Polymorphism database (dbSNP)) and prediction

tools (Sorting Intolerant from Tolerant (SIFT), Polymorphism Phenotyping (PolyPhen)). It

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depth.

Variant ranking

After using Polyweb filters to exclude a substantial amount of benign polymorphisms, several

candidate variants remained, requiring additional filtering strategies. The common

polymorphic variants that were frequently observed in the general population were excluded

using NGS population data bases (1000 Genomes Project, EVS, ExAC). In addition,

repositories of known disease-causing variants (dbSNP, ClinVar, OMIM) were used.

Moreover, prediction tools based on the analysis of the evolutionary conservation of the

nucleotide/amino acid targeted by the variant or on the prediction of the functional damage

caused by the mutated amino acid were used. Literature (e.g. PubMed, OMIM) was also used

to identify patients/families with similar phenotype carrying the same or other mutations in

the same gene, functional studies or to assess biological relevance. The above-mentioned

approaches aimed at reducing the number of variants of unknown significance (VUS)

identified, for whom available evidence is not conclusive to claim pathogenicity or to exclude

pathogenicity.

Exonic copy number variants (CNVs) detection pipeline

Putative heterozygous/homozygous/hemizygous structural variants or exonic CNVs were

highlighted using a method based on a double normalization of the average depth-of-

coverage, comparing the number of reads in the index sample with other regions in the same

sample and with the same region in the other samples.

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All candidate mutations were discussed collegially between geneticists and clinical

neurologists before validation. For the proof-of-principle experiment, identified variations

were systematically validated by Sanger sequencing in the 48 first patients. Sanger

sequencing has also been performed punctually, in particular to validate variants in

susceptibility or candidate genes, or CNVs. Moreover, three patients with molecularly

confirmed MD diagnosis, identified prior to this study by Sanger sequencing, were included

as control. In the event of VUS identification, its involvement in the patient’s phenotype was

investigated by specific imaging or biochemical tests if available or by segregation study

within the family of the patient.

Mutations were considered as causative if they fulfilled the following criteria: convincing

sequencing quality (high number of reads) occurring in genes which are consistent with the

phenotype, predicted to be deleterious by several algorithms, very rare, or absent in

population databases.

Whole-exome sequencing

Whole-exome sequencing was performed on DNA extracted from whole blood by exon

capture with the Agilent SureSelect kit and high-throughput sequencing with an Illumina

HiSeq2500 sequencer (IGBMC sequencing platform) for 13 individuals (WHA#, NLDT#,

ALG72) and at the Centre National de Génotypage, Institut de Génomique, CEA, France for

10 individuals (ATX#).

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Phenotype Name refSeq Disease Inheritance MIM Number Dementia, Lewy body 127750 AD SNCA including Rep1 NM_001146055 Parkinson disease 1 168601 AD promoter region Parkinson disease 4 605543 AD LRRK2 NM_198578 {Parkinson disease 8} 607060 AD 608013, 230800, Gaucher disease AR 230900, GBA NM_001005742 231000, 231005 {Lewy body dementia, susceptibility to} 127750 AD {Parkinson disease, late-onset, susceptibility to} 168600 AD PINK1 NM_032409 Parkinson disease 6, early onset 605909 AR DJ-1/PARK7 NM_007262 Parkinson disease 7, autosomal recessive early-onset 606324 AR ?Neurodegeneration with optic atrophy, childhood 615491 AR UCHL1 NM_004181 onset ?{Parkinson disease 5, susceptibility to} 613643 AD GIGYF2 NM_001103146 {Parkinson disease 11} 607688 AD parkin/PARK2 NM_004562 Parkinson disease, juvenile, type 2 600116 AR 3-methylglutaconic aciduria, type VIII 617248 AR HTRA2 NM_013247 {Parkinson disease 13} 610297 AD VPS35 NM_018206 {Parkinson disease 17} 614203 AD EIF4G1 NM_001194947 {Parkinson disease 18} 614251 AD Parkinson disease 19a, juvenile-onset 615528 AR DNAJC6 NM_001256864 Parkinson disease 19b, early-onset 615528 AR Reclassified - Variant of unknown significance DNAJC13 NM_015268 AD Parkinson disease 21 - AD SYNJ1/synaptojanin NM_003895 Parkinson disease 20, early-onset 615530 AR 1 RAB7L1 NM_001135662 {Parkinson disease 16} 613164 AD HARP syndrome 607236 AR PANK2 NM_153638 Neurodegeneration with brain iron accumulation 1 234200 AR Infantile neuroaxonal dystrophy 1 256600 AR PLA2G6 NM_003560 Neurodegeneration with brain iron accumulation 2B 610217 AR Parkinson disease 14, autosomal recessive 612953 AR ?Ceroid lipofuscinosis, neuronal, 12 606693 AR ATP13A2 NM_022089 Kufor-Rakeb syndrome 606693 AR SLC6A3 NM_001044 Parkinsonism-dystonia, infantile 613135 AR FBXO7 NM_012179 Parkinson disease 15, autosomal recessive 260300 AR Amyotrophic lateral sclerosis 5, juvenile 602099 AR SPG11 / spatacsin NM_025137 Spastic paraplegia 11, autosomal recessive 604360 AR SPG15 / ZFYVE26 NM_015346.3 Spastic paraplegia 15, autosomal recessive 270700 AR Mitochondrial DNA depletion syndrome 4A (Alpers 203700 AR type) Mitochondrial DNA depletion syndrome 4B (MNGIE POLG / POLG1 NM_001126131 613662 AR type) Mitochondrial recessive ataxia syndrome (includes 607459 AR SANDO and SCAE) Dementia, frontotemporal, with or without 600274 AD parkinsonism MAPT NM_001123066 Supranuclear palsy, progressive 601104 AD Supranuclear palsy, progressive atypical 260540 AR {Parkinson disease, susceptibility to} 168600 AD Ceroid lipofuscinosis, neuronal, 11 614706 AR GRN / progranulin NM_002087 Frontotemporal lobar degeneration with ubiquitin- 607485 AD positive inclusions DCTN1 / dynactin 1 NM_004082 Perry syndrome 168605 AD © 2018 American Medical Association. All rights reserved. Continued

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Phenotype Name refSeq Disease Inheritance MIM Number ATP6AP2 NM_005765 ?Parkinsonism with spasticity, X-linked 300911 AR HTT / huntingtin NM_002111 Huntington disease 143100 AD JPH1 / junctophilin NM_020647 Huntington-disease like NR NR 1 VPS13A NM_033305 Choreoacanthocytosis 200150 AR McLeod syndrome with or without chronic XK NM_021083 300842 XL granulomatous disease Chorea, hereditary benign 118700 AD NKX2-1 / TITF1 NM_001079668 Choreoathetosis, hypothyroidism, and neonatal 610978 AD respiratory distress ATP7B NM_000053 Wilson disease 277900 AR FTL NM_000146 Neurodegeneration with brain iron accumulation 3 606159 AD FA2H NM_024306 Spastic paraplegia 35, autosomal recessive 612319 AR WDR45 NM_001029896 Neurodegeneration with brain iron accumulation 5 300894 XLD Cerebellar ataxia 604290 AR CP / NM_000096 Hemosiderosis, systemic, due to aceruloplasminemia 604290 AR ceruleoplasmin [Hypoceruloplasminemia, hereditary] 604290 AR ?Spastic paraplegia 43, autosomal recessive 615043 AR C19orf12 NM_001031726 Neurodegeneration with brain iron accumulation 4 614298 AR DCAF17 / C2orf37 NM_025000 Woodhouse-Sakati syndrome 241080 AR COASY NM_001042532 Neurodegeneration with brain iron accumulation 6 615643 AR Creutzfeldt-Jakob disease 123400 AD PRNP / prion NM_001271561 Gerstmann-Straussler disease 137440 AD protein Huntington disease-like 1 603218 AD Ceroid lipofuscinosis, neuronal, 2 204500 AR TPP1 / CLN2 NM_000391 Spinocerebellar ataxia, autosomal recessive 7 609270 AR

CLN3 NM_001042432 Ceroid lipofuscinosis, neuronal, 3 204200 AR GM2-gangliosidosis, several forms 272800 AR HEXA NM_000520 Tay-Sachs disease 272800 AR HEXB NM_000521 Sandhoff disease, infantile, juvenile, and adult forms 268800 AR Dystonia, DOPA-responsive, with or without GCH1 NM_000161 128230 AD, AR hyperphenylalaninemia TH NM_199292 Segawa syndrome, recessive 605407 AR Dystonia, dopa-responsive, due to sepiapterin reductase SPR NM_003124 612716 ?AD, AR deficiency NPC1 NM_000271 Niemann-Pick disease, type C1 and D 257220 AR NPC2 NM_006432 Niemann-pick disease, type C2 607625 AR TIMM8A / DDP NM_004085 Mohr-Tranebjaerg syndrome 304700 XLR Mitochondrial DNA depletion syndrome 5 SUCLA2 NM_003850 (encephalomyopathic with or without methylmalonic 612073 AR aciduria) GM2A NM_000405 GM2-gangliosidosis, AB variant 272750 AR 230500, GM1-gangliosidosis, type I, II and III AR GLB1 NM_000404 230600, 230650 Mucopolysaccharidosis type IVB (Morquio) 253010 AR TOR1A / torsin 1A NM_000113 Dystonia-1, torsion 128100 AD TAF1 NM_004606 Dystonia-Parkinsonism, X-linked 314250 XLR TUBB4A / tubulin Dystonia 4, torsion, autosomal dominant 128101 AD NM_006087 beta 4A Leukodystrophy, hypomyelinating, 6 612438 AD SGCE / epsilon NM_001099400 Dystonia-11, myoclonic 159900 AD sarcoglycan THAP1 NM_018105 Dystonia 6, torsion 602629 AD PRKRA NM_003690 Dystonia 16 612067 AR SLC6A3 / DAT NM_001044 Parkinsonism-dystonia, infantile 613135 AR CIZ1 NM_001257975 ?Dystonia 23 614860 AD © 2018 American Medical Association. All rights reserved. Continued

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Phenotype Name refSeq Disease Inheritance MIM Number ANO3 / anoctamin 3 NM_031418 Dystonia 24 615034 AD GNAL NM_182978 Dystonia 25 615073 AD ATM NM_000051 Ataxia-telangiectasia 208900 AR HPRT1 NM_000194 Lesch-Nyhan syndrome 300322 XLR GCDH NM_000159 Glutaricaciduria, type I 231670 AR NM_017547, NM_002488, NM_005002, FOXRED1, NDUFA2, NM_004544, NDUFA9, NDUFA10, NM_018838, NDUFA12, NDUFAF2, NM_174889, Leigh syndrome due to mitochondrial complex I 256000 Mi, AR, XL NDUFAF6/c8orf38, NM_152416, deficiency NDUFS3, NDUFS4, NM_004551, NDUFS7, NDUFS8 NM_002495, NM_024407, NM_002496

Leigh syndrome 256000 Mi, AR SDHA NM_004168 Mitochondrial respiratory chain complex II deficiency 252011 AR DLD NM_000108 Dihydrolipoamide dehydrogenase deficiency 246900 AR SURF1 / surfeit 1 NM_003172 Leigh syndrome, due to COX IV deficiency 256000 Mi, AR Leigh syndrome due to cytochrome c oxidase COX15 NM_078470 256000 Mi, AR deficiency Thiamine metabolism dysfunction syndrome 2 (biotin- SLC19A3 NM_025243 607483 AR or thiamine-responsive encephalopathy type 2) Dystonia, primary cervical DRD5 NM_000798 {Blepharospasm, primary benign} 606798 IC ACTB / beta actin NM_001101 ?Dystonia, juvenile-onset 607371 AD ETHE1 NM_014297 Ethylmalonic encephalopathy 602473 AR Angiopathy, hereditary, with nephropathy, aneurysms, 611773 AD and muscle cramps COL4A1 NM_001845 Brain small vessel disease with or without ocular 607595 AD anomalies Porencephaly 1 175780 AD FUCA1 / alpha L NM_000147 Fucosidosis 230000 AR fucosidase GAMT NM_138924 Cerebral creatine deficiency syndrome 2 612736 AR Leukoencephalopathy with dystonia and motor SCP2 NM_002979 613724 AR neuropathy AP4B1 NM_001253852 Spastic paraplegia 47, autosomal recessive 614066 AR PRKCG NM_002739 Spinocerebellar ataxia 14 605361 AD ALDH5A1 NM_170740 Succinic semialdehyde dehydrogenase deficiency 271980 AR SUOX NM_001032387 Sulfite oxidase deficiency 272300 AR Ataxia, early-onset, with oculomotor apraxia and APTX / aprataxin NM_001195248 208920 AR hypoalbuminemia SETX / senataxin NM_015046 Spinocerebellar ataxia, autosomal recessive 1 606002 AR ARSA / arylsulfatase A NM_001085425 Metachromatic leukodystrophy 250100 AR GALC / NM_000153 Krabbe disease 245200 AR galactosylceramidase ABCD1 NM_000033 Adrenoleukodystrophy, adult adrenomyeloneuropathy 300100 XLR MMADHC / c2orf25 NM_015702 Methylmalonic aciduria and homocystinuria, cblD type 277410 AR 3-methylglutaconic aciduria with deafness, SERAC1 NM_032861 614739 AR encephalopathy, and Leigh-like syndrome SDHAF1 NM_001042631 Mitochondrial complex II deficiency 252011 AR ARX© 2018 AmericanNM_139058 Medical Association. All rights Partingtonreserved. syndrome 309510 XLR Pelizaeus-Merzbacher disease 312080 ContinuedXLR PLP1 NM_000533 Spastic paraplegia 2, X-linked 312920 XLR Downloaded From: https://jamanetwork.com/ on 10/02/2021 eTable 1. Continued

Phenotype MIM Name refSeq Disease Inheritance Number SEPSECS NM_016955 Pontocerebellar hypoplasia type 2D 613811 AR ALDH7A1 NM_001201377 Epilepsy, pyridoxine-dependent 266100 AR MECP2 NM_001110792 Rett syndrome 312750 XLD Convulsions, familial infantile, with paroxysmal 602066 AD PRRT2 NM_001256442 choreoathetosis Episodic kinesigenic dyskinesia 1 128200 AD Dystonia 9 601042 AD SLC2A1 / GLUT1 NM_006516 GLUT1 deficiency syndrome 1, infantile onset, severe 606777 AD, AR GLUT1 deficiency syndrome 2, childhood onset 612126 AD PNKD / MR1 / NM_015488 Paroxysmal nonkinesigenic dyskinesia 118800 AD DYT8 Alternating hemiplegia of childhood 2 614820 AD ATP1A3 NM_152296 CAPOS syndrome 601338 AD Dystonia-12 128235 AD KCNMA1 NM_001161352 Generalized epilepsy and paroxysmal dyskinesia 609446 AD NOL3 NM_001185057 Myoclonus, familial cortical 614937 AD ADCY5 NM_183357 Dyskinesia, familial, with facial myokymia 606703 AD SLC16A2 / MCT8 NM_006517 Allan-Herndon-Dudley syndrome 300523 XLD Episodic ataxia, type 2 108500 AD Migraine, familial hemiplegic, 1, with or without CACNA1A NM_023035 141500 AD progressive cerebellar ataxia Spinocerebellar ataxia 6 183086 AD KCNA1 NM_000217 Episodic ataxia/myokymia syndrome 160120 AD CACNB4 NM_000726 Episodic ataxia, type 5 613855 AD SLC1A3 NM_004172 Episodic ataxia, type 6 612656 AD PDHX NM_003477 Lacticacidemia due to PDX1 deficiency 245349 AR PDHA1 NM_001173454 Pyruvate dehydrogenase E1-alpha deficiency 312170 XLD Hyperekplexia, hereditary 1, autosomal dominant or GLRA1 NM_001146040 149400 AD, AR recessive GLRB NM_000824 Hyperekplexia 2, autosomal recessive 614619 AR COMT NM_000754 COMT-activity polymorphism NR NR SLC30A10 NM_018713 Hypermanganesemia with dystonia 1 613280 AR FOXG1 NM_005249 Rett syndrome, congenital variant 613454 IC RAB39B NM_171998 ?Waisman syndrome 311510 XLR

AD: autosomal dominant, AR: autosomal recessive, XLD: X-linked dominant, XLR: X-linked

recessive, Mi: mitochondrial, {}: incomplete penetrance, ?: debated, NR: not reported.

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2: eTable NLDT14 NLDT16 NLDT17 NLDT15 NLDT30 ATX167 Patient NLDT9 ALG72 WHA9 WHA7 ID

Gender Probable pathogenic variants identified in a cohort of 23 patients with cerebellar ataxia using WES analysis. WES using ataxia cerebellar with patients 23 of a cohort in identified variants pathogenic Probable M M M M M M F F F F

1 affected brother affected 1 1 affected brother brother affected 1 1 affected brother brother affected 1 1 affected brother brother affected 1 1 affected brother brother affected 1 2 affected sisters affected 2 1 affected uncle 1 affected 1 affected sister sister affected 1 1 Family history Family affected sister affected and sister sister and Sporadic

Strabismus symptom Primary Ataxia Ataxia Ataxia Ataxia Ataxia Ataxia Ataxia Ataxia Ataxia

Childhood (years) AAO 10 14 10 37 4 3 3 3 2

neuropathy with axonal involvement, deafness, Areflexia, babinski sign, apallesthesia, clubfoot, clubfoot, apallesthesia, sign, babinski Areflexia, dysmetria, dysmetria, demyelinating peripheralsensory neuropathy, Dysarthria, oculomotor apraxia, nystagmus, Distal weakness, mild demyelinating motor motor demyelinating mild weakness, Distal Oculomotor apraxia, clubfoot, axonal and disturbance, dysmetria, adiadochokinesia Hypotonia, ataxia,Hypotonia, nystagmus, dysarthria, deafness, tremor, clubfoot, deep sensory sensory deep clubfoot, tremor, deafness, Retinitis pigmentosa, cerebellar ataxia, ataxia, cerebellar pigmentosa, Retinitis Sensory neuropathy, deafness, altered Cerebellar ataxia, tetrapyramidal signs Sensorimotor axonal polyneuropathy adiadochokinesia,intention tremor Axonal polyneuropathy cerebellar atrophy cerebellar electroretinogram Additional signs Additional and dysarthria. optic atrophy Babinski sign Babinski nystagmus

HSD17B4 SLC52A2 SLC52A2 SLC52A2 PEX10 Gene Gene SPG7 APTX PEX6 SACS SACS SETX

inheritance Mode of of Mode AR AR AR AR AR AR AR AR AR AR

c.188 c.12220G>C; p.A4074P c.12220G>C; c.11707C>T; p.R3903* c.11707C>T; c.1529C>T; p.A510V / p.A510V c.1529C>T; c.1749G>C; p.W583C c.1749G>C; c.3967C>T; p.R1323* c.3967C>T; c.827G>T; p.C276F / / p.C276F c.827G>T; c.2735C>T; p.A912V c.2735C>T; c.916G>A; p.G306R p.G306R c.916G>A; c.932G>A; p.R311G c.932G>A; c.401C>T; p.P134L c.401C>T; c.837G>A; W279* c.837G>A; - 1G>A; M63_D99del M63_D99del 1G>A; / c.1444A>T Mutation(s)

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3: eTable Patient ID NLDT11 ATX534 ATX372 ATX853 ATX250 ATX599 ATX517 ATX476 ATX593 NLDT5 NLDT1 NLDT2 ATX52

Negative cases Negative Gender M M M F F F F F F F F F F

1 affected sister, ancient ancient sister, affected 1 Sporadic, consanguinity Sporadic, Sporadic, consanguinity Sporadic,

1 affected brother, 1 1 brother, affected 1 in a cohort of 23 patients with cerebellar ataxia using WES analysis. WES using ataxia cerebellar with patients 23 of a cohort in 2 affected affected 2 2 affected brothers affected 2 1 affected brother, brother, affected 1 brother, affected 1 affected sister, sister, affected Family History Family consanguinity consanguinity consanguinity consanguinity consanguinity Sporadic Sporadic Sporadic Sporadic Sporadic brothers, brothers,

Symptom Primary Ataxia Ataxia Ataxia Ataxia Ataxia Ataxia Ataxia Ataxia Ataxia Ataxia Ataxia Ataxia Ataxia

AAO (years) infancy 11 35 13 14 27 13 1 1 1 7 4 2

Cerebellar hypoplasia, ophthalmoparesis, enophthalmia, scoliosis, retarded growth, Cerebellar ataxia, dysarthria, adiadochokinesia, pyramidal syndrome, brisk reflexes, Gait ataxia, dysmetria, positive Romberg sign, absent deep tendon reflexes tendon deep absent sign, Romberg positive dysmetria, ataxia, Gait Cerebellar and pyramidal syndrome, sensorimotor axonal neuropathy Spastic ataxia, visual impairment, vesico impairment, visual ataxia, Spastic Head titubation, dysarthria, mild intellectual disability, epilepsy disability, intellectual mild dysarthria, titubation, Head Ataxia, sensorimotor neuropathy, hypoacousia, visual loss Congenital cerebellar ataxia, intellectual disability intellectual ataxia, cerebellar Congenital Ataxia, dystonia, spasticity, intellectual disability intellectual spasticity, dystonia, Ataxia, Cerebellar syndrome, bilateral visual blurring visual bilateral syndrome, Cerebellar Spastic ataxia with brain iron accumulation iron brain with ataxia Spastic Cerebellar syndrome, dystonia, tremor dystonia, syndrome, Cerebellar

Ataxia, sensory neuropathy dystonia, encephalopathy telangiectasia Phenotype

sphincterian disturbances

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Sporadic Mutation(s) Patient (S) / AAO Mode of Gender MDs Gene dbSNP/ ID Familial (years) inheritance Nomenclature Polyphen SIFT AF in ExAC (F) ClinVar PMD84 F F park 17 TAF1 XL D23G; htz NR 0,07 0,01 0 PMD90 M S dyt 22 MECP2 XL E19K; hemz NR 0,08 0,08 0 PMD91 F F dyt 26 DCTN1 AD S958A; htz NR 0,016 0,47 0 PMD102 M S dyt 16 RAB7L1 AD Q104*; htz NR - - 0 PMD104 M S park 20 SLC16A2 XL V218I; ho NR 0,998 0,15 0 PMD109 M F chorea 26 DCTN1 AD A1132G; htz NR 0,183 0,07 0 PMD121 F ND parox MD 16 FOXG1 AD H33R; htz NR - 0,31 0 GCH1 AD K54N; htz NR 0,004 0,17 0 PMD124 F S dyt 31 DCTN1 AD P209S; htz NR 0.003 0.52 0 PMD126 M S dyt 6 POLG AR/AD M1057T; htz NR 0.975 - 0 PMD129 M S myo 5 PRRT2 AD Y393H; htz NR - - 0 ATP1A3 AD I463T; htz NR 1 0.34 0 PMD137 M S dyt 6 c.1delA; htz NR 0 0 0 CP AR S14G; htz rs149864882 0.005 0.39 5.79e-05 CACNB4 AD c.-73delT; htz NR - - 0 PMD140 F S dyt 3 JPH1 AD S99N; htz NR 0.11 0.16 0 PMD151 F S park 35 CIZ1 AD I238V; htz NR 0.007 0.52 0 PMD159 F S dyt 48 TAF1 XL Q1687Q; htz NR 0 0 1.14e-05 Q226H; htz rs147282197 0.001 0.2 0.000399 PMD178 M F park 52 POLG AR/AD D930Y; htz NR 0.996 - 0 PMD182 M S chorea ND EIF4G1 AD Q595R; htz NR 0.147 0.41 8.243e-06 PMD187 F F dyt 40 XK XL A135V; htz NR 1 - 2.506e-05 PMD189 M S park 36 THAP1 AD K26M; htz NR 0.005 0.51 0 PMD191 F S dyt 5 LRRK2 AD L2002F; htz NR 1 0.01 0 PMD197 F ND chorea 50 EIF4G1 AD P269P; htz NR - - 0 PMD212 M F dyt 49 DNAJC13 AD V322L; htz NR 0.643 0.02 0 PMD213 F S park 36 DCTN1 AD R11W; htz NR 0.492 0.02 0 PMD217 F F park 25 SGCE AD E70D; htz NR 0.87 0.53 0 PMD218 M S park 43 DNAJC13 AD Q767H; htz NR 0.031 0.1 0 ADCY5 AD A350V; htz NR 0.742 0.06 0 PMD222 M F dyt ND LRRK2 AD M1702T; htz NR 0.989 - 1.649e-05 ANO3 AD CA/-; htz NR - - 0 PMD225 M ND park 56 CACNA1A AD A2447T; htz NR - 0.61 0

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Sporadic Mutation(s) Patient (S) / AAO Mode of Gender MDs Gene dbSNP/ ID Familial (years) inheritance Nomenclature Polyphen SIFT AF in ExAC (F) ClinVar c.3533-3C>T; PMD226 M F park 41 ADCY5 AD NR - - 0 htz PMD232 F ND park 35 GIGYF2 AD E428G; htz rs148790724 - 0.3 8.238e-06 PMD239 M ND park 58 GIGYF2 AD Q1017H; htz NR 0.801 0.18 0 PMD243 M ND myo 7 GBA AR/AD G416S; htz rs121908311 0.575 0.02 8.245e-06 PMD249 M S myo 1 ADCY5 AD L820V; htz NR 0.186 0.03 0 c.1398+5G>T; PMD257 M S myo 0 SLC6A3 AR NR - - 0 ho Y267N; htz rs781326840 0.006 0.32 0 PMD260 F F myo 14 SGCE AD c.390+64delGT; NR - - 0 htz PMD267 F F dyt 21 JPH1 AD c.216G>A; htz NR - - 0 PMD269 M S dyt 17 ABCD1 XL Q472P; hemz NR 0.078 0.12 0 PMD277 F F park 57 SLC1A3 AD I174T; htz NR 0.899 0.05 0

PMD278 F F park ND LRRK2 AD D996G; htz NR 0.528 0.09 0 PMD310 F F dyt ND SETX AR/AD T2279A; htz NR 0.996 0.03 0 PMD333 F S chorea 55 ANO3 AD G567R; htz NR 0.367 - 0 PMD336 M ND park ND COL4A1 AD P530S; htz rs145172612 0.005 0.06 0.0004129 PMD339 M ND park ND KCNMA1 AD K211E; htz NR 0.013 0.98 0 PMD345 M S park 35 GNAL AD D374E; htz NR 0.985 0.21 0 PMD348 F S dyt 3 PRRT2 AD A330T; htz NR 0.704 0.16 8.545e-06

PMD352 M S park 36 ADCY5 AD Y337N; htz NR 0.962 - 0 PMD353 F S park 68 GBA AR/AD L483P; htz rs421016 0.998 - 0.003099 PMD357 F F park 41 LRRK2 AD E1301K; htz NR 0.089 0.06 0 MAPT AD G536S; htz NR 1 0.04 1.778e-05 PMD366 F S park 52 SLC1A3 AD F178V; htz NR 0.872 0.14 0 G74V; htz NR 0.977 - 0 PMD375 F F park 48 GBA AR/AD c.221_222insT; NR 0 0 0 htz HTT AD A2560P; htz NR 0.739 0.19 0 PMD384 M F park 75 c.651+4insAAC; COL4A1 AD NR - - 0 htz PMD388 M ND park 4 LRRK2 AD A1880V; htz NR 0.437 0.17 0

PMD405 F F park 47 CIZ1 AD V407L; htz NR 0.001 0.5 0 PMD408 M S chorea 39 CACNA1A AD N1043K; htz NR 0.017 0.2 0 ANO3 AD R945Q; htz NR 0.999 0.47 0 PMD410 F F park 53 deletion of LRRK2 AD exons 48-51; NR - - - htz

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Sporadic Mutation(s) Patient (S) / AAO Mode of Gender MDs Gene dbSNP/ ID Familial (years) inheritance Nomenclature Polyphen SIFT AF in ExAC (F) ClinVar PMD411 M S park 54 PLP1 XL F27S; hemz NR 0.13 0.49 0 P1074L; htz rs780992643 0.907 - 0 ATP13A2 AR PMD418 F S park 52 R1158C; htz rs201610681 0.587 - 0.0004837 CIZ1 AD R891C; htz rs373808543 1 - 0

PMD420 M S parox MD 23 GBA AR/AD E365K; htz rs2230288 0.006 0.75 0.009792 PMD421 M F park ND EIF4G1 AD T404A; htz NR 0.067 0.79 0 PMD424 M ND dyt 53 LRRK2 AD Q923H; htz rs58559150 0.847 0.05 0.000151 PMD428 F S park ND SPR AD A82T; htz NR 0.013 0.15 0.0001012 PMD429 F S park 31 LRRK2 AD K1539E; htz rs199777744 0.008 0.23 8.276e-06

AAO: age at onset, ND: not done, MD: movement disorders, AF: allele frequency, F: female,

M: male, NR: not reported, park: parkinsonism, dyt: dystonia, myo: myoclonus, parox MD:

paroxysmal movement disorders, XL: X-linked, AD: autosomal dominant, AR: autosomal

recessive.

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eFigure 1. Examples of pedigrees of cases with identified causative variants.

Pedigrees of patient PMD38 (arrow) presenting early-onset dystonia-parkinsonism due to

compound heterozygous variants in the PARKIN gene (A) and in patient PMD279 (arrow)

presenting a classical Parkinson’s disease phenotype with a heterozygous variant in GBA (B).

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A B

eFigure 2. Pedigrees suggesting mendelian inheritance in negative cases.

Pedigrees suggesting autosomal dominant inheritance of dopa-responsive dystonia in patient PMD66 (arrow) (A) and in patient PMD93 (arrow) (B), and autosomal recessive inheritance of parkinsonism associated to pyramidal signs, cerebellar ataxia and dystonia in patient PMD147 (arrow) (C). No pathogenic variants were found in these families.

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eFigure 3. Examples of pedigrees of cases with identified VUSs.

Pedigrees suggesting a de novo mutation responsible for generalized dystonia in patient

PMD90 (arrow) (A) and autosomal recessive inheritance in patient PMD91 (arrow) (B).

VUSs were found in these patients.

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Peculiar clinical findings

Parkinsonism

A similar phenotype has been described due to somatic mosaicism in surviving males, to

germline or somatic mutations in females, as well as to skewing of X chromosome

inactivation. One patient with early-onset Parkinson’s disease (AAO 30 years) carried

pathogenic variants in two distinct MD-associated genes: a homozygous pathogenic variant

Q456* in PINK1, and a heterozygous pathogenic variant G2019S in LRRK2. In this case, the

PINK1 variant is more probable to be causative, given the young age at onset and the

incomplete penetrance of mutations in LRRK2 although whether the LRRK2 variant

contributed to the phenotype could be discussed.

Dystonia

Among 33 patients with focal cervical dystonia, a diagnosis was found in only 2, which is

significantly lower than in the other patients of the cohort (6%, 95% CI 0.02 to 0.86; P=.02).

One of these patients had a heterozygous duplication of exon 4 in TUBB4A, in which only

missense mutations were identified until yet in patients with a more severe phenotype

(“whispering” dysphonia and lower limb involvement). The second patient harbored a GCH1

variant affecting a splice site consistent with dopa-responsive dystonia (DYT5). Low doses of

levodopa were subsequently started leading to the rapid disappearance of the cervical

dystonia.

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