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RORB mutations in epilepsy

SUPPLEMENTARY Data for

Loss-of-function of the Retinoid-related nuclear receptor (RORB) gene and

epilepsy

Gabrielle Rudolf¶*1,2,3, Gaetan Lesca¶4,5,6, Mana M. Mehrjouy7, Audrey Labalme4, Manal

Salmi8,9,10, Iben Bache7,11, Nadine Bruneau8,9,10, Manuela Pendziwiat12, Joel Fluss13,

Julitta de Bellescize14, Julia Scholly3, Rikke S. Møller15,16, Dana Craiu17, Niels

Tommerup7, Maria Paola Valenti-Hirsch3, Caroline Schluth-Bolard4,5,6, Frédérique

Sloan-Béna18, Katherine L. Helbig19, Sarah Weckhuysen20, 21,22, Patrick Edery4,5,6, Safia

Coulbaut23, Mohamed Abbas23, Ingrid E. Scheffer24, Sha Tang19, Candace T. Myers25,

Hannah Stamberger20,21,22, Gemma L. Carvill25, Deepali N. Shinde19, Heather C.

Mefford25, Elena Neagu26, Robert Huether27, Hsiao-Mei Lu27, Alice Dica17, Julie S.

Cohen28, Catrinel Iliescu17, Cristina Pomeran17, James Rubenstein28,29, Ingo Helbig12,30,

Damien Sanlaville4,5,6, Edouard Hirsch1,2,3, Pierre Szepetowski*8,9,10.

1IGBMC, CNRS UMR7104, INSERM U964, Strasbourg University, France

2Federation of Translational Medicine, Strasbourg, France

3Department of Neurology, Strasbourg University Hospital, France

4Department of Genetics, Lyon University Hospitals, Lyon, France

5Claude Bernard Lyon I University, Lyon, France

6Lyon Neuroscience Research Centre, CNRS UMR5292, INSERM U1028, Lyon,

France

7Department of Cellular and Molecular Medicine, Wilhelm Johannsen Centre for

Functional Genome Research, University of Copenhagen, Copenhagen, Denmark

8INSERM U901, Marseille, France

9UMR S901, Aix-Marseille University, Marseille, France

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10Mediterranean Institute of Neurobiology (INMED), Marseille, France

11Department of Clinical Genetics, Copenhagen University Hospital Rigshospitalet,

Copenhagen, Denmark

12Department of Neuropediatrics, Christian-Albrechts-University of Kiel and University

Medical Center Schleswig-Holstein (UKSH), Kiel, Germany

13Pediatric Neurology, Child and Adolescent Department, Geneva University Hospitals,

Geneva, Switzerland

14Epilepsy, Sleep and Pediatric Neurophysiology Department, Lyon University

Hospitals, Lyon, France

15Danish Epilepsy Centre, Dianalund, Denmark

16Institute for Regional Health Research, University of Southern Denmark, Odense,

Denmark

17“Carol Davila” University of Medicine Bucharest, Department of Clinical

Neurosciences (No.6), Pediatric Neurology Clinic, Alexandru Obregia Hospital,

Bucharest, Romania

18Department of Medical Genetics, University Hospitals of Geneva, Geneva,

Switzerland

19Division of Clinical Genomics, Ambry Genetics, Aliso Viejo, CA, USA

20Neurogenetics Group, Department of Molecular Genetics, VIB, Antwerp, Belgium

21Laboratory of Neurogenetics, Institute Born-Bunge, University of Antwerp, Antwerp,

Belgium

22Division of Neurology, University Hospital Antwerp (UZA), Antwerp, Belgium.

23UCB-Pharma, Colombes, France

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24Florey Institute, University of Melbourne, Austin Health and Royal Children’s

Hospital, Melbourne, Australia

25Department of Pediatrics, Division of Genetic Medicine, University of Washington,

Seattle, USA

26Human Genetics Laboratory, “Mina Minovici” National Institute of Forensic

Medicine, Bucharest, Romania

27Department of Bioinformatics, Ambry Genetics, Aliso Viejo, CA, USA

28Department of Neurology and Developmental Medicine, Division of Neurogenetics,

Kennedy Krieger Institute, Baltimore, Maryland, USA

29Departments of Neurology and Pediatrics, Johns Hopkins University School of

Medicine, Baltimore, Maryland, USA

30Division of Neurology, The Children’s Hospital of Philadelphia, Philadelphia,

Pennsylvania, USA

¶Both authors contributed equally to this work

*To whom correspondence should be addressed:

Gabrielle Rudolf, Service de Neurologie, Hôpital de Hautepierre, 1 Avenue Molière

67098 Strasbourg cedex France, Tel.: +33 (0)3 8812 8639 - Fax : +33 (0)3 8812 8533 -

e-mail: [email protected]

or

Pierre Szepetowski, Mediterranean Institute of Neurobiology (INMED), Inserm

UMR_S901, Parc Scientifique de Luminy, BP 13, 13273 Marseille Cedex 09, Tel.: +33

(0)4 9182 8111 - Fax : +33 (0)4 9182 8101 - e-mail: [email protected]

SUPPLEMENTARY NOTE

Clinical data

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mailto:[email protected]


Patients with de novo mutations involving RORB

Patient AG1

This girl is the child of unrelated parents. The patient was born at 38 weeks with a birth

weight of 2,600g. Pregnancy, delivery, and neonatal period were unremarkable. She had

a positive family history for febrile seizures, which were present in the patient’s brother,

father, and paternal uncle.

She had her first seizure at the age of 3 years in the setting of fever. Seizures recurred

on the next day and were reportedly generalized tonic-clonic seizures lasting for 1-2

minutes. After the initial presentation, she continued to have febrile and afebrile

generalized tonic-clonic seizures until the age of 5 years, when seizures were eventually

controlled on medication. She was initially treated with valproic acid, which was

eventually switched to carbamazepine due to lack of seizure control. At the age of

inclusion (age 18), she has been seizure-free off medication for several years. The

patient had unremarkable routine and long-term EEGs during the initial presentation.

An EEG at the age of 4 years demonstrated some degree of non-specific background

slowing, but no epileptiform activity.

She had an unremarkable early development until the age of 15 months, when her

development plateaued and regressed in social and language domains. She subsequently

developed prominent stereotypies, compulsive behaviors, and aggression. She had

episodes of rapid behavioral cycling with disruptive behavior and pica and was given

the diagnosis of autism spectrum disorder at the age of 2 years. For behavioral issues,

she was treated with clonidine and risperidone. On formal neuropsychological testing,

she was found to have moderate to severe ID with a non-verbal IQ of 42, a verbal IQ of

43, and a full-scale IQ of 40. At the age of 18, the patient had some basic

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communication skills. She had profound sleep problems, which resulted in medication

with melatonin, trazodone, and gabapentin. Brain MRI at the age of 4 years was

unremarkable.

Diagnostic family-based exome sequencing was performed (trio exome sequencing) and

a de novo c.218T>C (p.Leu73Pro) mutation was identified. This mutation was

predicted to interfere with DNA binding of the RORB protein (Supplementary Figure

2A).

Patient RO1

This boy was the first child of non-consanguineous Caucasian parents. Pregnancy,

delivery and perinatal period were unremarkable. He was born at 38 weeks gestation

with Apgars 10/10/10. Seizures started at the age of 4 months with brief bilateral clonic

movements or the arms and legs and fixed gaze. EEG recordings during this time

showed a normal EEG background with bilateral spontaneous spike- and poly-spike

wave discharges lasting for up to 2 seconds, which occurred for up to 70 times per day.

EEG features and unremarkable development at this point, the patient was diagnosed

with Benign Myoclonic Epilepsy of Infancy (BMEI). At the age of one year, the seizure

type changed to daily brief tonic seizures, which then transitioned into clonic-atonic

seizures, resulting in frequent falls. He also had atypical absences. He never had

generalized tonic-clonic seizures. Seizures were not provoked by fever or hot

temperature, but he had frequent seizures induced by startles such as sudden noises. He

was treated with valproic acid, levetiracetam, lamotrigine, topiramate, clobazam,

clonazepam, and rufinamide without effect on the seizure burden. EEG in later

childhood showed an EEG background slow for age and frequent multifocal spikes.

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Development was unremarkable until the age of 10 months, when his development

slowed. At the age of four years, he was found to be at the level of a two year-old child

(developmental quotient of 50%) and was diagnosed with severe ID. He was found to

be hyperkinetic and erratic when walking, which resulted in him bumping into walls

and doors. He did not demonstrate focal neurological features and there was no truncal

or appendicular ataxia. MRIs were unremarkable at the ages of 5 months, 2.5 years, and

3 years.

He was found to have a de novo c.1249_1251delACG (p.Thr417del) in-frame deletion,

which was predicted to interfere with proper RORB cofactor binding resulting in a

disruption to DNA ligand binding (Supplementary Figure 2 B).

Patients with de novo microdeletions and balanced translocation involving RORB

Case n°9A1117

This 25-year-old woman was the first child of healthy unrelated parents. Her younger

sister was healthy. A maternal uncle and two of her first cousins had GTCS in

adulthood. Neonatal history and motor development were normal. At age three years,

speech delay and learning difficulties were apparent. She went to a special school.

Cognitive evaluation by WISC-III, at age 5.5 years, confirmed mild intellectual

impairment with significant dissociation between language (VIQ=46) and performance

skills (VIP=76). Between the ages of 5.5 and 6 years, she experienced three bilateral

clonic seizures during febrile episodes. Interictal EEG recording showed normal

background rhythm and bilateral centrotemporal spikes. She was treated with

Carbamazepine, Valproate, Ethosuximide, and Vigabatrin. At age 10 years, EEG

recording revealed typical 3-Hz spike and wave absence seizures, characterized by

complete impairment of consciousness, oral or limb automatisms, and was activated by

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hyperventilation. She was not photosensitive. Clobazam replaced Vigabatrin because of

its potentially aggravating effect. A 24-h ambulatory EEG recording demonstrated

persistent absence seizures, lasting 6-10 seconds and interictal generalized spike or poly

spike waves, lasting 0.5 to 3 seconds. Focal frontal or temporal occipital paroxysmal

activity with variable hemispheric localization was also present. Lamotrigine was

introduced with good efficacy. At age 13 years, three tonic-clonic seizures occurred on

awakening, precipitated by sleep deprivation. In the following year, four more

convulsive seizures occurred, starting with absences or absence status with eyeball

elevation. Absence status lasted 30 minutes for one episode and she had rare eyelid

myoclonia for another despite changing to Topiramate. Since then, seizure freedom has

been obtained by Levetiracetam. At 25 years of age, cranial circumference was 57 cm

(+2.5 SD) and she was overweight (+4 SD) for a normal height (+0.5 SD). She had no

dysmorphic features. She had a modified half time job in a supermarket. Brain MRI was

normal.

Standard karyotype on 50 mitoses revealed a complex mosaic chromosomes imbalances

composed of a small supernumerary ring marker chromosome in 25 mitoses (50%), a

derivative chromosome 9 from a 9;21 translocation in one mitose (2%), and a normal

46,XX population in 24 mitoses (48%): mos 47,XX,+r[20]/46,XX,-21,+der(9)t(9;21)

[5]/46,XX[25]. In addition, aCGH analysis revealed a homogeneous 8.5-Mb deletion of

the long arm of chromosome 9 (chr9:g.(70984481_79549501)del, containing 47 genes

including RORB and a mosaic gain involving the whole short arm of chromosome 9

(Fig 2A). FISH analysis confirmed the homogeneous 9q13q21.13 deletion with the

RP11-404E6 BAC clone. It also showed an isochromosome 9p in 7% of cells, the

derivative 9 der(9)t(9;21) in 8% of cells and a small supernumerary ring chromosome 9,

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mainly composed of heterochromatin in 23% of cells (Fig 3) . All these rearrangements

were absent in the parents’ blood samples.

Case n°GE0705

This 10-year-old female patient was the second child of healthy unrelated parents. There

was no family history of seizures. The pregnancy was unremarkable. She presented with

brief absence seizures at the age of 4 years 9 months. EEG recordings showed absence

seizures lasting 5-10 seconds, occasionally with eyelid myoclonia, and triggered by IPS.

Ethosuximide was efficacious but poorly tolerated. The treatment was changed to

sodium valproate, which was not effective, then to Lamotrigine and Levitiracetam,

which had greater efficacy although seizures persisted. At the age of 10 years, she

presented with her first nocturnal generalized tonic clonic seizure. A nocturnal EEG was

performed that displayed significant worsening with multiple generalized 3-Hz

discharges, most often without clinical manifestation, occurring in the wake and sleep

state. She is currently on Lamotrigine and Levetiracetam and a ketogenic diet has just

been started. She displayed global developmental delay with early learning difficulties

and predominant speech impairment. At the age of 9 years her IQ was < 50. She has a

convergent strabismus and hypermetropia but otherwise her clinical examination is

unremarkable. Brain magnetic resonance imaging (MRI) and magnetic resonance

spectroscopy (MRS) were normal.

Array-CGH analysis showed a homogeneous 52-kb microdeletion of exons 5 to 10 of

RORB (chr9: g.(77261322_77313598del) (Fig 2B)

Case n°DK8393

This 10 year-old female is the first child of healthy non consanguineous parents. There

was no family history of seizures but the mother has had 5 spontaneous abortions.

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Pregnancy was without complications and she was born at gestational week 42 by

caesarian section because of mal presentation, with a birth weight of 4,000g, length 55

cm and head circumference 34 cm with full Apgar score. The patient was seen for

neuropediatric evaluation at 8 months of age because of delayed motor development and

poor eye contact. Examination showed hypermobility and general hypotonia. No facial

dysmorphic features or congenital malformations were found. Weight: 10 kg (+1SD),

height 74.5 cm (+1 SD) and head circumference 45.5 cm (+1 SD). Brain MRI and EEG

were performed at the age of 10 and 18 month respectively: both examinations were

normal. Ophthalmological examination was normal except from discrete intermittent

strabismus.

At age 3.5 years, the patient was tested by Bayley III and Vineland Adaptive Behavior

Scales and found to have moderate ID and infantile autism. She has attended care and

school for children with special needs since the age of 4.

She currently has severely delayed language development using single words, sounds

and pictures for communication. She is assisted for many of her daily needs and is

living in a residence for children and adolescents with ID or autism spectrum disorders.

She is very sensitive toward noise. From early childhood she has slept 7 hours or less

per night with more pronounced sleep disturbances during the winter time. A trial of

Melatonin resulted in psychological distress without beneficial effect on her sleep.

Seizures have never been observed. She has no dysmorphic facial features. She is 130

cm tall (- 2 SD) and her weight is 29 kg (- 2 SD).

Array-CGH analysis was normal. Conventional cytogenetic analysis revealed a de novo

balanced reciprocal translocation, t(9;19)(q21;q12)dn. The breakpoints were mapped by

mate-pair sequencing: out of 34,071,005 read pairs passing the alignment score, 19

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discordant mate-pair reads defined the breakpoints to chr9:g.77174312_77175018

(hg19), within the first intron of RORB (Fig 4A,B), and chr19: g.31139347_31139735,

truncating transcript ENST00000592773, distal to the annotated part of ZNF536. Sanger

sequencing refined the breakpoints to chr19: g.31139486_31139488, with a single

nucleotide (T) deletion (nt 31139487), and to chr9: g.77174984, with two small

duplications of chr9:77174985-77174991 and chr9:77174993-77174996 (Fig 4C). The

final karyotype according to the proposed ISCN/HGVS nomenclature is:

seq[GRCh37] t(9;19)(q21;q12)dn g.[chr9:pter_cen_77174984::chr19:31139488_qter]

g.

[chr19:pter_cen_31139486::chr9:77174985_77174991dup::chr9:77174993_77174996d

up_qter]

Case EC-CAE300

This patient is the child of non-consanguineous Caucasian parents. Pregnancy was

unremarkable. He was born at 36 weeks gestational age through an emergency C-

section due to uterine rupture. He did not have dysmorphic features. Generalized tonic-

clonic seizures started at the age of 2-3 years. He was initially started on valproic acid,

which controlled the seizures and could be weaned in the interim. At the age of 13

years, nocturnal generalized tonic-clonic seizures recurred and valproate was started

again. Carbamazepine was added due to insufficient seizure control, but showed no

additional effect. Levetiracetam monotherapy was started subsequently. This controlled

seizures, but led to serious behavioral side effects. Valproic acid monotherapy

eventually, led to good seizure control at the time of the last visit at the age of 28 years.

In addition to the generalized tonic-clonic seizures, he had absence seizures during

childhood. EEGs were unremarkable at the age of 13 and 16 years, and showed

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paroxysmal generalized slow-spike wave activity at age 19. Brain MRI was normal at

the age of 13 and 16 years. He has some behavioral problems with aggressive features

and is known to “act out”. During childhood, sleeping problems had been reported. He

has learning disabilities, and has a diagnosis of dyslexia, but official neuropsychological

testing has not been performed. Affymetrix SNP 6.0 array showed a microdeletion

including the RORB gene (chr9:g.76601085_77182821 del), which was validated with

an in-house developed technique for Multiplex Amplicon Quantification (MAQ). The

variant was not present in the patient’s mother. Paternal DNA was not available for

testing.

SUPPLEMENTARY METHODS

Functional studies: RNAs, cDNA synthesis

RNAs were extracted from EBV-immortalized lymphoblastoid cell lines established

from two patients of family 1 (patients 4 and 13) and from one unaffected relative

(family member n°16) using the RNeasy minikit (Qiagen, Courtaboeuf, France). Each

sample was treated with or without puromycin in order to evaluate the potential effect of

NMD (Non-sense mediated mRNA decay) on the mutant transcripts. Reverse-

transcription was performed using the Expand RT kit (Roche Applied Science, Meylan,

France). PCR was performed with a pair of primers located in exon 2 and 4,

respectively. PCR products were purified and analyzed by Sanger sequencing.

Cell culture transfection assays

RORB cDNA construct for the expression of either of RORβ fusion wild-type or mutant

protein with a FLAG at the N-terminus were transfected in COS-7 cells and detected

using anti-FLAG antibody.

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Constructs and site-directed mutagenesis

The RORB cDNA construct (Genecopoeia EX-Z5728-M11) was used for expression of

the corresponding wild-type human protein (Genbank NP_008845) with a FLAG at the

N-terminus. Quick-Change Site-Directed Mutagenesis kit (Agilent Technologies) was

used to generate the EX-Z5728-M11-Arg66* vector allowing the expression of the

mutant forms (p.Arg66*) of ROR-beta. The presence of the desired variant and the

absence of any other unwanted variant were confirmed by direct sequencing (GATC

Biotech).

Cell cultures, transfections and immunocytochemistry experiments

Monkey kidney fibroblast like cell line COS-7 cells (African green monkey kidney

fibroblast-like cell line established from CV-1 cells; ATCC CRL-1651) were seeded in

six-well plates with a concentration of 105 cells/well one day before transfection.

Transfections were accomplished by addition of 4µg of plasmid DNA and 12 µl

lipofectamine 2000 and 4µl of combiMag (OzBiosciences, France). FLAG-tag fusion

protein ROR-beta was detected using an antibody to the N-terminal FLAG (1:50,

Sigma). Nuclei were counterstained with DAPI (4’,6-diamidino-2-phenylindole).

Immunocytochemistry images were captured with a fluorescence microscope (Zeiss

Axio Imager Z2) with ApoTome attachment.

Structural modelling of p.L73P and p.T417del variants in RORβ

Ligand regulated transcription factors (LRTF) participate in many basic biological

processes and observed alterations, have been associated with many types of diseases1.

A subfamily of LRTF, the retinoic acid receptor-related orphan receptors (RORs), is

under active investigation as they have been implicated in many autoimmune type

diseases1. We have observed one missense, and one in-frame deletion (p.L73 and

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p.T417del) in RORβ. Structural investigation localizes the p.L73P variant in the DNA

binding domain and p.T417del in the ligand binding domain (LBD) of RORβ.

Although different in their mode of disruption, both variants result in an inactive RORβ

protein.

Structurally, Leu 73 occurs in the RORβ N-terminal DNA binding domain on a helix

directed towards the core of the DNA binding residues (Fig 2A). The helix acts to

directly stabilize one Zn binding site and support a second Zn binding site (labelled Zn1

and Zn2 in Supplementary Fig 2A). The hydrophobic Leu73 is partially buried and

interacts with the surrounding sidechains of Lys11 (K11), Ile12 (I12), and Val24 (V24).

The variant Leu 73 Pro (p.L73P) introduces a radically different backbone atom

arrangements resulting in severe steric constraints on the DNA binding domain

(Supplementary Fig 2B). The calculated energy change is significantly destabilizing

(5.2 ±1.3 kcal/mol) which is well beyond pathogenic cutoffs (1.6 kcal/mol) used in

RASopathy proteins2. The p.L73P variant likely introduces a destabilizing effect

resulting in a loss of structure and binding to RORβ DNA targets.

RORβ activation and suppression is controlled by corepressors, coactivators, and

ligands3. These molecules target, bind and alter the C-terminal ligand binding domain

(LBD). We observed a p.T417 in-frame deletion in the LBD domain of RORβ. The

alteration occurs within a ligand binding amphipathic helix near the coactivator binding

site (Supplementary Fig 2C). Thr417 participates in stabilization of the tertiary

structure by interacting with Glu312 (E312: Supplementary Fig 2C) on an adjacent

helix. Loss of a residue within the helix would cause a shift in the

hydrophobic/hydrophilic register likely resulting in an increase in instability. The exact

molecular consequences are unknown but we speculate the in-frame deletion results in a

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destabilized helix impairing either natural ligand or coactivator binding. This is

supported by the observation that the ligand binding domain is unstable in the absence

of cofactors4. Lack of binding or loss of correct dynamics within the protein would

result in RORβ inhibition and loss of binding DNA targets.

We observed germline variants p.L73P and p.T417 in-frame deletion in the DNA and

Ligand binding domains of RORβ respectively. Each disrupts the protein in a unique

way. However the end result is the same, anticipated loss of RORβ DNA binding.

Methods.

Structure of N-terminal DNA binding domain of RORβ (residues 45-97) was modelled

with ROSETTA35 using chain A (42% identity) from the structure of nuclear receptor

retinoid X receptor (residues 160-211 PDB: 3DZU)6. Structure of RORβ c-terminal

ligand binding domain (PDB: 1N4H) was used for analysis3. Energy calculations were

performed using FoldX7 in triplicate and all visualizations were performed using

PyMol8.

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http://www.ncbi.nlm.nih.gov/pubmed/?term=Trucks%20H%5BAuthor%5D&cauthor=true&cauthor_uid=25950944

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ruppert%20AK%5BAuthor%5D&cauthor=true&cauthor_uid=25950944

http://www.ncbi.nlm.nih.gov/pubmed/?term=Lal%20D%5BAuthor%5D&cauthor=true&cauthor_uid=25950944


SUPPLEMENTARY Figure 1. Cell culture transfection assays

No nucleoplasmic detection of p.Arg66* RORβ mutant protein in vitro.

COS-7 cells were transfected with either of wild-type (wt) or mutant (p.Arg66*)

constructs for the expression of the corresponding fusion proteins with a FLAG at the

N-terminus. (A) RORB transcripts corresponding to the wt or to the mutant constructs

were detected by RT-PCR from RNAs extracted from transfected COS-7 cells. NT:

non-transfected cells. (B) GAPDH primers were used as controls. (C-E) The RORB-wt

fusion protein was detected in the nuclei of transfected cells, as expected. (F-H) In

contrast, no nucleoplasmic fluorescence was detected with the mutant RORβ-Arg66*

fusion protein. Fusion proteins were detected with anti-FLAG antibodies (red). DAPI:

nuclear staining (blue). Bar: 10 µm

SUPPLEMENTARY Figure 2. Structural modelling of the de novo RORB

mutations

A) Cartoon structure of wild type RORβ N-terminal DNA binding domain (green)

modelled with DNA (orange cartoon) from PDB:3DZU. Zinc molecules are labelled as

ZN1/ZN2 and modelled as gray spheres. Leu 73 is shown as magenta stick. Selected

residues with 6 Å shown as sticks and labeled with gray text. B) RORβ N-terminal

DNA binding domain with modeled Pro73 as a white stick. Steric clashes are indicated

by an arrow and shown as red disks. C) Cartoon structure of RORβ C-terminal ligand

binding domain (green) with bound Retinoic Acid (orange stick labelled “REA”).

Thr417 is show as magenta stick with the interaction to E312 shown as dotted black

line. Selected residues with 6 Å shown as sticks and labeled with gray text.

SUPPLEMENTARY Figure 3. RORB promoter structure

16

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378


The importance of the RORB-promoter region illustrated by its high conservation,

embedment within a >16kb transposon-free region (blue box in the Repeating Elements

track), with several CpG-islands and multiple transcription factor binding sites.

SUPPLEMENTARY Figure 4. Patient GE0705 qPCR data

Analysis done by quantitative PCR SybrGreen with 2 different assays, designed in the

RORB gene (assay 1 and 2). The results confirmed, in patient GE0705, the occurrence

of an heterozygous deletion in the RORB gene. The parental samples (mother and

father) show two normal copies and do not carry the deletion as the reference control.

SUPPLEMENTARY Table 1. Summary of clinical and genetic data from all cases and

family with RORB (NM_006914.3) defects and epilepsy.

17

379

380

381

382

383

384

385

386

387

388


Patients Gender Age at onset (y.m)

Type of seizure

EEG PhotoS IQ Additional features

Gene variant/CNV Ref

4 F 13 Absences with eyelid myoclonia, GTCS (13 to 31 y.)

3 Hz generalized SW

yes 62 (VIQ 51, PIQ 83)

- c.196C>T (p.(Arg66*) this study

10* F - - Slow wave during IPS

yes NA - c.196C>T (p.(Arg66*) this study

13 F 3 Absences with eyelid myoclonia, GTCS

3 Hz generalized SW

yes 62 (VIQ 51, PIQ 83)


14 F 9 Absences with eyelid myoclonia

3 Hz generalized SW

yes 73 (VIQ 63, PIQ 89)


20 F 11 Absences with eyelid myoclonia, GTCS

3 Hz generalized SW

yes NA - c.196C>T (p.(Arg66*) this study

23 F 9 Reported with one episode of absence seizure

NA NA NA - c.196C>T (p.(Arg66*) this study

18


patient AG1

F 3 Febrile and afebrile GTCS

Normal No moderate to severe ID (FSIQ 40)

autism spectrum; obsessive compulsive disorder; aggression; anxiety; severe sleep disorder from early childhood

de novoc.218T>C p.(Leu73Pro)

this study

patient RO1

M 4 myoclonic, tonic, clonic, atonic, atypical absence

polyspike-wave provoked by IPS; multifocal spikes

yes severe ID hyperkinetic with no clear ataxia

de novo c.1249_1251delACG (p.(Thr417del))

this study

patient 9A1117

F

5.5

CS (5.5 y.), absences with rare eyelid myoclonia (10 y.), GTCS and absence status (13 y.)

CTS (5.5 y.), 3 Hz generalized SW (10 y.)

Nomild ID (VIQ 46, PIQ 76)

-de novo(chr9:g.70984481_79549501 del)

this study

patient GE0705

F4.9

Absences with occasional

3 Hz generalized SW

yes IQ < 50Strabismus, predominant speech delay

de novo(chr9:g.77261322_77313598 del)

this study

19


eyelid myoclonia

patient DK8393

F

0.8 no seizures Normal no Mdr-svr ID

autism spectrum disorder; severe sleep disorder from early childhood

de novo seq[GRch37]t(9;19)(q21;q12)dn this study

patient CE-CAE300

M 2 Nocturnal GTCS, absence seizures starting at 10 y

Generalized slow-spike wave

NA Learning disability, IQ testing not performed

Sleeping problems, agressive behaviour

(chr9:g.76601085_77182821 del)

9 and this study

Patient BL1

M

5

Hypotonic seizure without abnormal movements

Interictal grapho-elements with left central and right occipital predominance

NA svr ID

Autistic behavior, microcephaly, mild facial dysmorphism

de novo (chr9:g.73920064_79528981 del) 10 ≠

Patient BL3

M

5 Absence NA NA mdr ID

Autistic behavior, growth delay, moderate facial dysmorphism

de novo (chr9:g.72182945_79312316 del) 10 ≠

Patient BL5

M4 Occipital

lobe Posterior and generalized yes mld ID Mild facial

dysmorphismde novo(chr9:g. 76998441_79292639 10 ≠

20


epilepsy (4 to 5 y.), GTCS (10 years)

SW del)

Patient BL6

F

3.5

Febrile and afebrile GTCS (3.5 y.)

SW over the right temporal region

NA IQ < 50 Dysmorphic features

de novo (chr9:g.74391462_85348850 del) 10 ≠

Patient BL7

M

5.5 Atypical absences

SW over the left occipital or parietal region with continuous firing during sleep

NA IQ 47 Short stature

de novo(chr9: g.77047592_79291317 del) 10 ≠

Patient BL8

F

13

GTCS, absences and myoclonia

Rhythmic spike and SW activity at 2.5 - 3 Hz

NA mdr-svr

Autistic behavior, facial dysmorphism, short stature

de novo (chr9:g.71025186_77807150 del) 10 ≠

Patient 12

M

2Eyelid myoclonia and GTCS

CTS NA NA Autism

de novo (chr9:g.73931220_76496752 del) 11

Patient 1

F

4.5SGE with absences and GTCS

Left CTS NAmild ID (VIQ 65, PIQ 80)

dysmorphic features

de novo (chr9:g.77283017_78323705 del) 12

21

389


CNV: copy number variation; CS: clonic seizures; CTS: centro-temporal spike; del:

deletion; EEG: electroencephalogram; GTCS: generalized tonic clonic seizures; Hz:

hertz;g ID: intellectual disability; IPS: intermittent photic stimulation; IQ: intellectual

quotient; kb: kilobases; Mb: megabases; mdr: moderate; mld: mild; NA: not available;

PhotoS: photosensitivity; PIQ: performance IQ; Ref: reference; SGE: secondarily

generalized seizures; svr: severe; SW: spike and wave discharges; VIQ: verbal IQ; y.:

years. *Patient 10 in family 1 had photosensitivity at time of inclusion but seizure state

could not be proven; she was included in this table as she inherited the p.Arg66* RORB

variant. ≠ Decipher data. For the sake of clarity, cases that have been reported

previously are italicized.

SUPPLEMENTARY Table 2. Summary of whole exome sequencing data obtained in

Family 1.

(A) List of the missense and splice-site variants found by exome sequencing and shared

by three patients with GGE and eyelid myoclonia in family 1 (i.e. as detected in family

members n°4, 13 and 20) (Fig 1A).

(B) List of the missense and splice-site variants shared by four family members with

photoparoxysmal response (PPR), regardless of their epileptic state (i.e. as detected in

family members n°4, 13, 20 and 21).

Genomic position are given (nt, nucleotides) according to the human genome reference

sequence hg19.

22

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409


chromosome (chr)

position (nt)

gene Genbank refseq protein or gene variation

Occurrence in the ExAC database

A

chr1 22903330 EPHA8 NM_020526NM_00100694

C260WC260W

0

chr1 109265134 FNDC7 NM_00114493 G259V 72

chr1 152185632 HRNR NM_00100993 G2825S 5

chr2 225684224 DOCK10 NM_01468 R1069Q 0

chr3 45718415 LIMD1 NM_014240 D632V 0

chr3 64526863 ADAMTS9 NM_18292 R1810H 73

chr5 10402505 MARCH6 NM_00588 T355A 0

chr5 13759007 DNAH5 NM_00136 A3456V 56

chr5 68728878 MARVELD2 NM_001038603 D487E 0

chr5 140573461 PCDHB10 NM_01893 D446N 0

chr6 31974850 CYP21A2 NM_000500NM_00112859

GAG/-GAG/-GAG/-

9

chr6 35258039 ZNF76 NM_003427.4 c.433-4C>G 531

chr6 90428706 MDN1 NM_01461 P2034L 5

chr6 139564226 TXLNB NM_15323 T498P 79

chr6 158994513 TMEM181 NM_02082 F161V 42

chr7 17379269 AHR NM_00162 L607P 3

chr7 103138569 RELN NM_173054NM_00504

T2933IT2933I

27

chr7 111629112 DOCK4 NM_01470 D141G 3

chr7 127961330 RBM28 NM_018077 R518C 248

23


NM_00116613 R377C

chr7 139762578 PARP12 NM_02275 P24A 17

chr8 61769019 CHD7 NM_01778 L2394F 0

chr9 15744329 C9orf93 NM_17355 H703R 10

chr9 77249649 RORB NM_006914 R66* 0

chr9 109691447 ZNF462 NM_02122 D1752N 64

chr9 117169021 DFNB31 NM_001083885NM_015404NM_00117342

S234LS617LS617L

26

chr11 373809 B4GALNT4 NM_17853 S255L 11

chr11 14666116 PDE3B NM_00092 D165E 573

chr11 118851363 FOXR1 NM_18172 R259C 5

chr11 126176484 DCPS NM_01402 D74G 21

chr11 134031768 NCAPD3 NM_01526 I1198V 1

chr13 32972347 BRCA2 NM_00005 TGTA/-TGTA/-TGTA/-

12

chr13 95131389 DCT NM_001922NM_00112988

C41RC41R

1

chr13 95363512 SOX21 NM_00708 Q264H 0

chr13 96515937 UGGT2 NM_02012 D1197V 56

chr13 114303693 ATP4B NM_00070 K291M 72

chr14 39788463 CTAGE5 NM_203356NM_001247989NM_005930NM_203354NM_001247990NM_203355NM_00124798

A475VA509VA504VA492VA429VA504VA475V

2

chr15 41191918 VPS18 NM_02085 R301L 8

24


chr15 42148796 SPTBN5 NM_01664 N2902Y 366

chr15 42378436 PLA2G4D NM_17803 R121Q 65

chr15 44067735 ELL3 NM_02516 Q186P 2

chr15 75942740 SNX33 NM_15327 P433T 2

chr16 57723714 GPR97 ENST0000032765 G304G 69

chr16 71724422 PHLPP2 NM_015020ENST00000299971ENST0000036042

K203KK10KK203K

0

chr16 81201642 PKD1L2 NM_05289 R992H 5

chr16 81211460 PKD1L2 NM_001076780NM_05289

A797TA797T

1

chr17 19318492 RNF112 NM_00714 T423M 1

chr17 38064467 GSDMB NM_001165959NM_00116595

N221SN221S

4

chr17 40183643 ZNF385C ENST00000436535NM_00124270

S224GS145G

0

chr17 64731691 PRKCA NM_00273 V381M 10

chr20 48522841 SPATA2 NM_001135773NM_00603

R293HR293H

286

chr22 24373140 LOC391322 NM_00114493 M1I 1

chrX 107819161 COL4A5 NM_000495NM_03338

I190VI190V

4

B

chr1 109265134 FNDC7 NM_00114493 G259V 72

chr3 45718415 LIMD1 NM_01424D632V

0

chr3 64526863 ADAMTS9 NM_18292 R1810H 73

chr5 68728878 MARVELD2 ENST00000413223 D371E 025


NM_001038603NM_001244734ENST0000045429

D487ED475ED475E

chr6 90428706 MDN1 NM_01461 P2034L 5

chr7 17379269 AHR NM_00162 L607P 3

chr7 103138569 RELN NM_173054NM_00504

T2933IT2933I

27

chr7 111629112 DOCK4 NM_01470 D141G 3

chr7 127961330 RBM28 NM_018077NM_00116613

R518CR377C

248

chr7 139762578 PARP12 NM_02275 P24A 17

chr13 96515937 UGGT2 NM_02012 D1197V 56

chr13 114303693 ATP4B NM_00070 K291M 72

chr14 39788463 CTAGE5 NM_203356NM_001247989NM_005930NM_203354NM_001247990NM_203355NM_00124798

A475VA509VA504VA492VA429VA504VA475V

2

chr17 64731691 PRKCA NM_00273 V381M 10

26

410

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