# RORA-neurodevelopmental disorder: A unique triad of developmental disabilities, cerebellar anomalies, and myoclonic seizures

Mariagrazia Talarico, Julitta de Bellescize, Matthias de Wachter, Xavier Le Guillou, Guylène Le Meur, Matthieu Egloff, Bertrand Isidor, Benjamin Cogné, Diane Beysen, Paul Rollier, et al.

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Mariagrazia Talarico, Julitta de Bellescize, Matthias de Wachter, Xavier Le Guillou, Guylène Le Meur, et al. RORA-neurodevelopmental disorder: A unique triad of developmental disabilities, cerebellar anomalies, and myoclonic seizures. Genetics in Medicine, 2024, 27 (4), pp.101347. 10.1016/j.gim.2024.101347. hal-05037055

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ARTICLE

**RORA-neurodevelopmental disorder: A unique triad of
developmental disabilities, cerebellar anomalies, and
myoclonic seizures**

<figure><img src="image_2.png" /></figure>

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updates

Mariagrazia Talarico<sup>1,2,3 ID</sup>, Julitta de Bellescize<sup>4</sup>, Matthias De Wachter<sup>5</sup>,
Xavier Le Guillou<sup>6,7</sup>, Guylène Le Meur<sup>8</sup>, Matthieu Egloff<sup>6,7</sup>, Bertrand Isidor<sup>9,10</sup>,
Benjamin Cogné<sup>9,10</sup>, Diane Beysen<sup>5</sup>, Paul Rollier<sup>11</sup>, Melanie Fradin<sup>11</sup>, Laurent Pasquier<sup>11</sup>,
Ilaria Guella<sup>12</sup>, Scott E. Hickey<sup>13</sup>, Paul J. Benke<sup>14</sup>, Amelle Shillington<sup>15</sup>, Candy Kumps<sup>16</sup>,
Olivier Vanakker<sup>16</sup>, Erica H. Gerkes<sup>17</sup>, Shenela Lakhani<sup>18</sup>, Irina Romanova<sup>19,20</sup>,
Ilya Kanivets<sup>21</sup>, Melanie Brugger<sup>22,23</sup>, Katharina Vill<sup>22,24</sup>, Raymond C. Caylor<sup>25</sup>,
Cindy Skinner<sup>25</sup>, Rory J. Tinker<sup>26</sup>, Tommy Stödberg<sup>27</sup>, Astrid Nümann<sup>28</sup>, Tobias B. Haack<sup>29</sup>,
Natalie Deininger<sup>29</sup>, Holger Hengel<sup>29,30</sup>, Jeanne Jury<sup>9,10</sup>, Solène Conrad<sup>9,10</sup>,
Sandra Mercier<sup>9,10</sup>, Grace Yoon<sup>31</sup>, Melissa Tsuboyama<sup>32</sup>, Giulia Barcia<sup>33</sup>, Cyril Gitiaux<sup>33</sup>,
Marlène Rio<sup>33</sup>, Andrea Bevot<sup>34</sup>, Sylvia Redon<sup>35,36,37</sup>, Kevin Uguen<sup>35,36,37</sup>,
Antje Wonneberger<sup>38</sup>, Alexander Schulz<sup>39</sup>, Dagmar Timmann<sup>40</sup>, Danielle Hays Karlowicz<sup>41</sup>,
Nicolas Chatron<sup>2,3</sup>, Amanda Carnevale<sup>31</sup>, Sonal Mahida<sup>32</sup>, Katrin Õunap<sup>42,43</sup>,
Sébastien Kury<sup>9,10</sup>, Sara Cabet<sup>3,44</sup>, Gaetan Lesca<sup>2,3,* ID</sup>

ARTICLE INFO

Article history:

Received 28 June 2024
Received in revised form
10 December 2024
Accepted 11 December 2024
Available online 17 December 2024

Keywords:

Cerebellar hypoplasia
Developmental disorder
Myoclonic seizures
RORA
Vermian atrophy

ABSTRACT

**Purpose:** RORA encodes the RAR-related orphan receptor-α, playing a pivotal role in cerebellar maturation and function. Here, we report the largest series of individuals with RORA-related-neurodevelopmental disorder.

**Methods:** Forty individuals (30 unrelated; 10 siblings from 4 families) carrying RORA pathogenic/likely pathogenic variants were collected through an international collaboration.

**Results:** The 33 variants (29 de novo, 4 inherited, and 1 shared), identified by genome/exome sequencing (n = 21), chromosomal microarray analysis (n = 7), or gene panels (n = 4), included frameshift (n = 18/33), missense (n = 9/33), and stop codon (n = 6/33). Developmental disability (n = 32/37), intellectual disability (n = 22/32), and cerebellar signs (n = 25/34) were the most striking clinical features. Cerebellar symptoms were divided into early-onset, late-onset, and progressive subgroups. Cerebellar hypoplasia, atrophy, or both (n = 16/25) were more frequent in individuals with missense variants in the DNA-binding domain. Epilepsy (n = 18/38), with prominent myoclonic seizure types (n = 11/18), was classified in (1)

The Article Publishing Charge (APC) for this article was paid by the University Hospitals of Lyon (Hospices Civils de Lyon).

Mariagrazia Talarico, Julitta de Bellescize, Matthias De Wachter, Sébastien Kury, Sara Cabet, and Gaetan Lesca contributed equally to the work.

*Correspondence and requests for materials should be addressed to Gaetan Lesca, Service de Génétique, Groupement Hospitalier Est, 59 Boulevard Pinel, 69677 Bron, France. Email address: gaetan.lesca@chu-lyon.fr

Affiliations are at the end of the document.
genetic generalized epilepsy (n = 10/18) with a syndromic diagnosis identifiable for 6: epilepsy with eyelid myoclonia (n = 5/6) and epilepsy with myoclonic absence (n = 1/6); (2) developmental and epileptic encephalopathy (n = 5/18); and (3) unclassified (n = 3/18). A participant with rapid deterioration of visual acuity and cone/rod dystrophy was reported.

**Conclusion:** Missense variants in DNA-binding domain correlate to a more severe cerebellar phenotype. The RORA-related-neurodevelopmental disorder triad comprises developmental disability, cerebellar features, and a spectrum of myoclonic epilepsy.

© 2024 The Authors. Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/).

## Introduction

The RORA gene (HGNC:10258), located on human chromosome 15q22.2, encodes the retinoic-acid-receptor-related orphan receptor-α (RORα) that belongs to the highly conserved retinoid-related orphan nuclear receptor (NR) family, including also RORβ (RORB, HGNC:10259) and RORγ (RORC, HGNC:10260).¹ RORs are multifunctional transcription factors involved in the complex regulation of circadian rhythm, brain development, cellular metabolism, and immunity. They share a highly conserved structure consisting of a DNA-binding domain (DBD) and a Ligand-binding domain (LBD)²: the DBD binds to specific DNA sequences called ROR response elements adjacent to the promoter region of target genes. Characterizing endogenous ligands is still an ongoing challenge that could offer, together with the development of synthetic ligands, new therapeutic approaches in a variety of human diseases.³ Alternative splicing leads to 4 RORα isoforms (RORα1-4) that only differ in the N-terminus. In humans, they are widely expressed in various tissues with a pattern of tissue-specific expression: RORα3 is only present in the testis, whereas RORα1 and RORα4 are primarily expressed in the cerebellum, particularly in Purkinje cells (PCs), as well as in thalamic nuclei and retinal ganglion cells.³,⁴ The GTex portal (https://gtexportal.org/home/) report details the tissue-specific RORA gene expression in the human brain: primarily cerebellum, then amygdala, frontal cortex, caudate nucleus, cortex, frontal cortex, hippocampus, hypothalamus, nucleus accumbens, putamen, spinal cord (cervical c-1), and substantia nigra. The spontaneous mutant mouse staggerer (*Rora*<sup>sg/sg</sup>) and *Rora*<sup>-/-</sup> mice revealed the role of RORα in the maturation, physiological function, and anatomical integrity of the cerebellum from embryonic development to adulthood.³,⁵ Disruption of RORα in zebrafish larvae resulted similarly in cerebellar hypoplasia caused by loss of PCs and Granule cells.⁶ RORA polymorphisms were first suggested as rare susceptibility factors for epilepsy,⁷ and more recently, RORA was recognized as a target gene for autism spectrum disorder (ASD) by identifying reduced RORA expression in affected individuals and in their post-mortem cerebellum and frontal cortex.⁸ The first direct implication of RORA in

Mendelian diseases was demonstrated in 2018 by Guissart et al,⁶ who reported 16 RORA pathogenic variants in individuals with a significant association to developmental disability (DD), intellectual disability (ID), cerebellar symptoms, epilepsy, and ASD. In addition, experimental data on Zebrafish suggested that missense variants could have distinct pathogenic mechanisms of either haploinsufficiency or a dominant toxic effect due to their localization in the LBD or in the DBD, respectively.

In this study we report a large series of individuals with RORA-NDD, focusing on cerebellar and epileptic seizure phenotypes. In addition, we report a participant with early and progressive myopia with cone and rod dystrophy.

## Materials and Methods

### Participants' recruitment and phenotyping

Participants were recruited through the Matchmaker Exchange platform, the EUROEPINOMICS RES network, the French EPIGENE network,⁹ the NETRE network, and ClinVar database. All clinical, genetic, and additional data were collected through a standardized questionnaire. When available, seizure semiology, electroencephalography (EEG) recordings, cognitive findings, and brain magnetic resonance imaging (MRIs) were retrospectively analyzed by an experienced epileptologist (J.d.B.), a neuro-pediatrician (M.D.W), and a pediatric neuro-radiologist (S.C.). Seizure types and epilepsy syndromes were classified.¹⁰ The level of DD was evaluated by reporting milestones and by reporting the severity of ID. ID was classified in individuals aged 5 years and older, based on intellectual quotient (IQ) score as borderline (IQ 70-84), mild (IQ 50-55 to 69), moderate (IQ 35-40 to 50-55), severe (IQ 20-25 to 35-40), or profound (IQ below 20-25). If psychometric data were not available, classification was based on clinical grounds.¹¹

Ophthalmological data were evaluated by each inclusion center and reported in the standard survey. For 1 enrolled participant presenting a particular ophthalmological condition, the methodology of examination adopted by the inclusion center was reported in the Supplemental Methods.

All of the abbreviations are listed in the Supplemental Abbreviation List.
## Genetic analyses

Each inclusion center used the strategy and technology established by their standard laboratory procedures for genetic testing. RORA variants were identified through proband-only exome sequencing (n = 10/33, 30.3%), chromosomal microarray analysis (n = 7/33, 21.2%), proband-only genome sequencing (n = 5/33, 15.2%), trio-based-exome sequencing (n = 4/33, 12.1%), trio-based-genome sequencing (n = 2/33, 6.1%), trio-based analysis of an ASD gene panel (n = 1/33, 3%), proband-only epi-lepsy gene panel (n = 2/33, 6.1%), or proband-only ID gene panel (n = 1/33, 3%). The deletion identified in 2 siblings (32A and 32B) was confirmed in the unaffected mother (32C) by fluorescence in situ hybridization. Individual 16, for whom the RORA variant was identified with chromosomal microarray analysis, also underwent a retinopathy gene panel with negative results, and then an exome sequencing that did not reveal any significant results. Whenever DNA samples were available, Sanger sequencing was applied to evaluate the segregation of the variants in the families of index cases.

RORA variants (HGNC:10258) were reported according to the Human Genome Variation Society guidelines, based on the Genome Reference Consortium Human Reference sequence version 37 (GRCh37/hg19) and the RefSeq MANE-selected transcript NM_134261.3. The VariantVалиdator tool was applied for compliance with Human Genome Variation Society nomenclature.¹²

To evaluate the chromosomal deletions, the deleted regions were investigated through the UCSC Genome Browser (https://genome.ucsc.edu/) on GRCh37/hg19 human genome. The genes involved in the deletions, including RORA, were identified and were subjected to a meticulous check through the Online Mendelian Inheritance in Man (OMIM) database (https://omim.org/), to evaluate their possible involvement in determining the phenotypes.

To predict the consequences on protein function of missense variants, in silico tools were used, displayed on the MobiDetails interface (https://mobidetails.iurc.montp.inserm.fr/MD/): MutationTaster (https://www.mutationtaster.org/), Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT¹³, CADD¹⁴, Revel,¹⁵ and AlphaMissense (https://alphamissense.hegelab.org).¹⁶ The MetaDome interface was used to evaluate the regional protein intolerance to missense variants.¹⁷

Based on segregation analysis, in silico predictions and allele frequency in population database (Genome Aggregation Database; GnomAD v4.0.0, https://gnomad.broadinstitute.org/, Database of Genomic Variants, DGV http://dgv.tcg.ac.ca/dgv/app/home), the collected variants were classified by following the American College of Medical Genetics and Genomics and Genomics/Association for Molecular Pathology guidelines.¹⁸

## Statistical analysis

Genotype-phenotype correlation was investigated initially in this series of 40 RORA affected individuals, then in a combined series of 56 individuals, comprising those reported by Guissart et al⁶; it is noteworthy that additional information was provided for 3 individuals of the previous study.

To correlate the phenotype and variant type, we established 2 groups: (1) deletions (losses including RORA and in-frame deletions) and truncating variants (including frameshift and nonsense variants) and (2) missense variants.

To examine the impact of missense variant localization, (1) missense variants occurring in DBD were compared with missense variants occurring in LBD; (2) missense variants affecting DBD were compared with missense variants affecting all other regions of the gene and deletions.

All of these independent qualitative variables were compared using Fisher's exact test. To reduce false-positive errors, the Bonferroni correction was applied. The threshold considered statistically significant was P value < .05. Statistical analysis was done with the software “R” version 4.3.1 released on June 16, 2023.

## Standard protocol approvals, registrations, and participant consents

All parents or legal guardians of the affected individuals gave informed consent for inclusion. This study was approved by the local institutional Ethics Committee (Commission Nationale de l'Informatique et des Libertés Register Number: 22_5867). All data collected were handled in accordance with the “Directive on the protection of individuals with regard to the processing of personal data” and the national legislation applicable to it, as required by “The European General Data Protection Regulation” (General Data Protection Regulation [AVG/GDPR]-EU2016/679) and the French legislation elaborating on this regulation.

## Results

### Clinical features

We identified 40 unreported individuals with pathogenic and likely pathogenic RORA variants, including 10 affected members belonging to 4 unrelated families. Median age at inclusion was 8 years (Q1: 5.0, Q3: 22.0, range: 6 months-74 years). Twenty-two out of 40 individuals were male. The main clinical data and the amount of unavailable information are summarized in Figure 1 and in
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

**Figure 1** Clinical features. A. The histogram summarizes the clinical features observed in this series of 40 individuals. ADHD, attention deficit hyperactivity disorder; ASD, autism spectrum disorder; DD, developmental delay; ID, intellectual disability. B. The cake graphs show the distribution of epileptic syndromes and seizure types. DEE, developmental and epileptic encephalopathy; EEM, epilepsy with eyelid myoclonia; EMA, myoclonic absence epilepsy; EMAtS, epilepsy with myoclonic-atonic seizures; GGE, genetic generalized epilepsy; IESS, infantile epileptic spasm syndrome. C. EEG pattern of eyelid myoclonia: sharply contoured posterior activity in the alpha range at eye closure. Reference montage, 100 µV/mm. This EEG was recorded at the age of 15 of individual 18 (EEM syndrome).
**Supplemental Table 1.** In the following paragraphs, denominators indicate the number of individuals in whom the clinical information could be assessed.

## Neurodevelopmental features

DD occurred in 32 individuals (32/37, 86.5%). Normal motor developmental milestones were seen in 16 individuals (16/40, 40%) with a median age of walking at 18 months (Q1: 16, Q3: 24). Most individuals presented with speech delay (n = 25/33, 75.7%), 8 presented with normal speech development (8/33, 24.3%). The median age of the first words was 22 months (Q1: 16, Q3: 25). At inclusion, 2 individuals were not able to produce any words (n = 2/25, 8%). Verbal abilities in the remainder of individuals ranged from using a few words (n = 4/23, 17.4%) to age-appropriate speech at the last evaluation (n = 9/23, 39.1%). Eight individuals had speech dysarthria (n = 8/23, 34.8%). Developmental stagnation was observed in 5 (n = 5/32, 15.6%). Individuals 11, 25, 31, 32A, and 32B) and regression in 2 (n = 2/32, 6.2%. Individuals 15 and 17).

ID was present in 22 individuals (22/32, 68.8%. ID was not possible to assess for 7 individuals because of age), spanning from mild (n = 15/22, 68.2%), to moderate (5/22, 27.7%), and severe (2/22, 9.1%). Ten individuals had normal or borderline cognitive functioning.

Behavioral problems were described in 13 individuals (13/40, 32.5%) comprising anxiety (n = 4/13, 30.8%), aggression (n = 5/13, 38.5%) obsessive-compulsive behavior (n = 3/13, 23.1%), and chronic pain and non-epileptic seizures (n = 1/13, 7.8%). Individual 18 had depression with suicide attempt. Attention deficit hyperactivity disorder was present in 7 individuals (n = 7/30, 23.3%) and ASD, or features consistent with autism, was diagnosed in 7 participants (n = 7/34, 20.6%). Sleep disturbances, mostly night-time awakenings, were reported in 9 of 27 individuals (33.3%) and not evaluated in 13. Sleep apnea syndrome was not reported, and polysomnography was not performed.

## Neurological examination

Twenty-five individuals (n = 25/34, 73.5%) had at least 1 clinical cerebellar feature. Ataxia or unstable gait was present in 18 participants (n = 18/25, 72%). We could distinguish 2 groups according to the age at onset of ataxia: an early-onset group, usually diagnosed at walking, and a late-onset group, starting between 25 and 65 years. A global coordination disorder or at least impairment of fine motor skills was present in 13 individuals (n = 13/25, 52%). Dysmetria was noticed in 4 (n = 4/25, 16%), tremor in 6 (n = 6/25, 24%. One worsened with lamotrigine and another started with the introduction of valproic acid), and dysarthria in 5 (n = 5/25, 20%). Five individuals were presented with progressive cerebellar pathology (n = 5/25, 20%. Individuals 17, 21A, 22, 23, and 34A), and 2 of them were

wheelchair dependent. Eight individuals were reported as having oculomotor dyspraxia, a disturbance of saccadic eye movements or nystagmus (n = 8/25, 32%). Hypotonia was present in 10 individuals (n = 10/25, 40%), severe in 1 (n = 1/10, 10%), and not associated with other clinical or radiological cerebellar signs in only 2 individuals. Other neurological findings were rare: sensorimotor neuropathy (2), bilateral carpal tunnel syndrome (1), pyramidal signs (1), and idiopathic intracranial hypertension (1).

## Neuroimaging data

Brain MRI results were available for 30 individuals (n = 30/40, 75%), considered initially normal for 17 (n = 17/30, 56.7%) and showing mild nonspecific supratentorial anomalies in 3 (n = 3/30, 10%). For 16 of 30 (n = 16/30, 53.3%), cerebellar abnormalities were described. Reassessment of MRI files was possible for 15 (n = 15/30, 50%) and for case 2 of the former study. Eleven of the 15 reassessed MRI (11/15, 73.3%) displayed cerebellar hypoplasia predominant on the vermis (1/11, 9%), isolated vermian hypoplasia (3/11, 27.3%), vermian atrophy (5/11, 45.5%) often predominant on the upper part of the vermis, and association of cerebellar (2/11, 18.2%) or vermian (1/11, 9%) hypoplasia and atrophy. Both were present in case 2 of the former study, which showed a significant progression of cerebellar atrophy on successive MRIs (Figure 2). Serial MRIs were also performed in individual 28 and showed an appearance of vermian atrophy. Overall cerebellar pathology was present in at least 16 individuals (55%) after a reinterpretation of the MRI.

## Epilepsy

In this cohort, half of the individuals had epileptic seizures (18/38, 47%), individual 5 had a single febrile seizure at the age of 2, and seizure status was not known for 2 individuals. Median age of seizure onset was 3 years (Q1: 1.3; Q3: 6.50, range: 6 months-16 years). Seizures were mostly generalized at onset, as those appearing at evolution (14/18; 78%) and comprising myoclonic seizure forms (11/18, 61%). Status epilepticus occurred (6/18, 33%) and was nonconvulsive (n = 2), convulsive (n = 2), with myoclonic absences (n = 1), or myoclonic (n = 1). EEG data were available in 17 of 18 (94%). Background rhythm (BR) was normal (12/15; 80%), intermixed with beta in 1, slow in 2, and then slowed in 5. When sleep was recorded, physiological sleep features were present (11/12, 92%; not known for individual 10). Interictal paroxysmal discharges (14/17, 82%) were generalized (6/17, 35%), generalized and focal/multifocal (5/17, 29%) or focal/multifocal (3/17, 18%). Photosensitivity (5/13; 38%) and eye closure sensitivity (ecS) 4/13, 30%) were also assessed.

Epilepsy could be classified into 3 groups according to the classification of the International League Against Epilepsy¹⁰: developmental and epileptic encephalopathy (DEE, n = 5), genetic generalized epilepsy (GGE, n = 10), and unclassified (n = 3), Supplemental Table 2). In the DEE
<figure><img src="image_6.png" /></figure>

**Figure 2** MRI. Mid-sagittal brain MR images showing vermian hypoplasia in individual 3 (p.(Ala135Pro), [A]) and in individual 16 (deletion arr[hg19] 15q22.2 (61,487,264_61,696,482)x1, [B]), defined as a reduction in the size of the vermis with normal organization and trophicity. A vermian atrophy is observed in individual 21A (p.(Gly92Ser), [C]) and individual 25 (p.(Arg98Lys), [D]), predominant on the upper part of the vermis, with enlargement of the primary fissure (arrow) and defined as a loss of parenchyma and clearly visible foliation. Serial MRIs in individual 2 by Guissart et al⁶ showed initial cerebellar hypoplasia predominant on the vermis (p.(Gly92Ala), [E]) and, during follow-up, an appearance of cerebellar atrophy predominantly on the vermis (F) starting on the upper part of the vermis.

group, infantile epileptic spasms syndrome was diagnosed in 2 (individuals 9 and 17) with onset at 6 and 7 months, respectively, preceded in individual 17 by focal and generalized motor seizures. Three individuals had myoclonic epilepsy, starting between 6 and 9 months in 2 and at the age of 3 years with myoclonia and/or atonic or myoclonic-atonic seizure in 2. Epilepsy with myoclonic-atonic seizures (formerly known as Doose syndrome) was diagnosed in one (individual 3) and suspected in individual 10, but data were scarce. Seizure types in the GGE group comprised (1) eyelid myoclonia with absences (n = 5), combined with limb myoclonia in 4, (onset ranged between 1 year and 10

months and 9 years and 4 months), leading to the diagnosis of typical epilepsy with eyelid myoclonia (EEM, formerly known as Jeavons syndrome) in individual 18 and a EEM-like syndrome in 4; (2) myoclonic absences at age of 10 years in individual 27, who was diagnosed epilepsy with myoclonic absence; (3) absences (n = 2) with onset at 3 and 4.5 years; and (4) generalized tonic-clonic seizures (n = 2) with onset at 5 and 16 years, respectively. In these individuals, a specific epilepsy syndrome could not be established. In individual 12, absences at 3 Hz were very short. Childhood absence epilepsy was initially suspected in the individual 20; however, the onset was below the age of
4 years, and seizure control was incomplete in adolescence. In addition, flickering light was reported as triggering absences, photosensitivity and ecS were also present. The syndromic diagnosis for individual 4 with tonic-clonic seizures could be discussed as a single right frontal central seizure was recorded but without other arguments at evolution for focal epilepsy. The unclassified group (n = 3) had in common a complex association of motor (focal and/or bilateral) (and eventually impaired awareness) and myoclonic seizure types, beginning between 3 and 7 years. Interestingly, we reported the follow-up of case 6 from the former study: myoclonia occurred at evolution after initial multifocal seizures.

EEG in the nonseizure group when available (7/19; 37%) was normal (3/7), showed slowing (2/7), or showed inter-ictal discharges (2/7).

## Seizure/developmental outcome and antiseizure medication (ASM)

Preexisting DD was severe only in the DEE-infantile epileptic spasms syndrome individuals, with regression documented at least in individual 17. All individuals in this category were pharmacoresistant, similar to those in the unclassified group, but in the latter, neurodevelopmental features were less severe, having mild (n = 2) or moderate (n = 1) delay without regression. In the GGE group, rare, generalized tonic-clonic seizures appeared in 5 individuals. Seizure outcome was variable: 3 individuals were seizure free, similar to the updated case 13 from the former study, with a follow-up of 6 months to 3 years at the last assessment. In the 6 others, 1 to 4 appropriate ASM were used, of whom individual 20 is still under tri-therapy. Of interest, 3 were primarily seizure free under valproic acid or ethosuximide, then relapsed. Further documentation was not always satisfactory, but seizure frequency seemed to be improved. Individual 1 remained pharmacoresistant, and epilepsy-related factors could play a role in the reported learning difficulties. The preexisting developmental features in the GGE group were qualified as borderline (n = 2) or mild DD (n = 4) and were not known for 3. The unique case with developmental stagnation reported was unrelated to epilepsy factors because he had only a few tonic-clonic seizures between 5 and 7 years and was then seizure free under valproic acid monotherapy with a follow-up of 3 years at the last assessment. He had moderate DD. The most used drugs were valproic acid (n = 9), levetiracetam (n = 5), and ethosuximide (n = 4) followed by lamotrigine (n = 3), benzodiazepines (n = 3), topiramate (n = 2), and lacosamide (n = 2). Other ASMs were used, mostly in the DEE or unclassified group: zonisamide, cannabidiol, and methylprednisolone. Interestingly, oxcarbazepine triggered seizure exacerbation (individual 26). Ketogenic or a modified Atkins diet was started in 3 and was judged to be helpful in 2 (individuals 3 and 26), and 2 were on vagal nerve stimulators.

## Ophthalmological findings

In addition to the already-specified neurological eye movement disorder, 12 individuals had myopia, hypermetropia, or strabismus.

Individual 16, a 24-year-old woman, had the most severe ophthalmological findings, including early and progressive myopia, as well as cone and rod dystrophy (Figure 3). She wore glasses for high myopia (−10 D) since the age of 3. She had a decline in visual acuity from the age of 20 from 4/10 to 1/20 in a 4-year period. The macula appeared similar to a cherry red spot surrounded by gray coloring at the fundus (Figure 3A and A'), with hypoautofluorescence of the macula (Figure 3B and B'). The ellipsoid zone was disrupted in the fovea area (Figure 3C and C') and also in the peripheral retina in optic coherence tomography (Figure 3D and D'). The peripheral visual field was constricted (Figure 3E and 3E') and associated with a central scotoma (Figure 3F and F'). The electroretinogram was subnormal with focal central damage at the multifocal electroretinogram (Figure 3G and G').

## Dysmorphic features

For half of the individuals, minor dysmorphic facial features were reported by the clinician (19/40, 48%). They were highly variable from one patient to another, with no specific pattern, and therefore are not shown here. The following features were reported more than once: a small, sunken, or depressed nasal bridge (7), broad or high forehead (6), large tongue or tongue protrusion (3), triangular shaped face (3), simple ears with forward facing earlobe (3), blue sclerae (3), hypertelorism (2), long face (2), low-set ears (2), and posteriorly rotated ears (2). Microcephaly and macrocephaly were reported in 2 and 3 individuals, respectively, with only 1 having macrocephaly from birth. Short stature was reported in 3.

## Genetic findings

In this series of 40 individuals, we identified 33 hitherto unreported RORA variants (Figure 4, Supplemental Table 2). In 29 individuals, the variant occurred or was presumed to be de novo (29/40, 72.5%), in 10 it was inherited (10/40, 25%), showing incomplete penetration (9 out of 10 parents were healthy heterozygous for a pathogenic variant), and it was unknown in 1 (1/40, 2.5%).

The majority of variants are widely distributed along the messenger RNA sequence and cause frameshifts (18/33, 54.5%). They are specifically deletions (14/18, 77.7%) and frameshift insertions (4/18; 22.3%). Nine variants were missense (9/33, 27.3%), 5 affecting the DBD, 2 the LBD, 1 located just before the sequence encoding the DBD, and 1 just after the LBD. Six variants were nonsense (6/33, 18.2%), 1 occurred in DBD, 4 in the region between the
<figure><img src="image_7.png" /><figcaption>**Figure 3** Ophthalmological exams showing the cone rod dystrophy in individual 16. The macula appears cherry red spot in the fundus (Clarus Zeiss) (A and A') and hypoautofluorescent in autofluorescence exam (SPECTRALIS) (B and B'). The ellipsoid zone is also disrupted in the fovea area (C and C') and the peripheral retina (SPECTRALIS) (D and D'). The visual field is constricted with a central scotoma (E, F, E', and F'). The fovea peak is blunted at the multifocal ERG (G and G') (Roland). Right eye: (A, B, C, D, E, F, and G); left eye, (A', B', C', D', E', F', and G'). The * indicates the macula, and the arrow indicates the ellipsoid zone.</figcaption></figure>

DBD and LBD, and 1 in the last exon corresponding to the C terminus domain of the protein.

One variant, c.499C>T p.(Gln167Ter), was recurrent in 2 unrelated individuals (4, 30). Four variants, the missense c.274G>A p.(Gly92Ser), the microdeletions arr[hg19] 15q22.2 (60,886,678_61,161,374)x1 and arr[hg19]

15q22.2(60,768,415_60,956,191)x1, and the nonsense c.919G>T p.(Glu307Ter), were present in 2 or 3 siblings (families 21, 32, 33, and 34) and inherited from the mother in all cases. In family 33 the variant was proven de novo in the mother, whereas in the other families, it was not possible to assess. In family 34, the mother was affected as
<figure><img src="image_8.png" /><figcaption>**Figure 4** RORA variants in RORα protein representation. A. The figure shows the RORα protein representation, reporting all the identified variants in this series of 40 individuals (in black) and the single-nucleotide variations identified in individuals with follow-up from Guissart et al⁶ (in light blue), in their aminoacidic position. The variants located above the protein are frameshift and nonsense, whereas the variants located below the protein are missense. All variants are mentioned with their aminoacidic change (p.); then in brackets, the following are reported: the identification number of the individual, a black dot indicating epilepsy/epileptic seizures, a gray triangle indicating cerebellar signs, and a black star indicating ASD or autistic features. In position 92, 2 different missense variants were identified: p.(Gly92Ser) shared by 2 siblings (21A and 21B) and p.(Gly92Ala) identified in individual 22 of this case series and in individual 2 of the previous case series. p.(Gln167Ter) was identified in 2 unrelated individuals (4 and 30). p.(Glu307Ter) was identified in 2 siblings (34A and 34B). B. The color scale graph is the MetaDome representation of the protein, showing the position tolerance to the missense variants. Green and blue indicate the tolerance of the aminoacidic position to missense variants, yellow indicates the neutral tolerance of the aminoacidic position, and orange and red indicate the intolerance of the aminoacidic position to missense variants. On the horizontal gray Protein bar, the 2 binding domains are shown in pink (DBD on the left and LBD on the right) and the identified missense variants are represented by vertical green bars. The Amino acids and Exons black bars are reported in correspondence with protein figures on a dimensional scale. ASD, autism spectrum disorder; DBD, DNA-binding domain; LBD, Ligand-binding domain.</figcaption></figure>

well; in families 21, 32, and 33, the mother was unaffected.

Four chromosomal deletions, including the RORA gene, were reported. The arr[hg19] 15q21.3q22.31(54,879, 765_64,973,929)x1 involved the deletion of 63 coding genes, of which 23 OMIM genes were associated to a phenotype. The arr[hg19] 15q21.2q22.2(52,340,221_62, 790,591)x1 involved 51 coding genes, of which 22 were OMIM genes. The arr[hg19] 15q21.3q22.2(53,313,391_63, 042,697)x1 deleted 45 coding genes, of which 17 were OMIM genes. The arr[hg19] 15q21.3q22.2(55,083,180_62, 687,339)x1 involved 41 coding genes, of which 18 were OMIM genes. All the other OMIM genes involved in the

deletions were considered not relevant to phenotypic manifestations.

The 9 missense variants particularly drew our attention because they were mainly located in the functional domains of the protein. Of particular interest was the region of the DBD ranging from 90 to 100 amino acids, which was affected by 3 of the 9 missense variants. Three missense variants from the previous study also affected this region.⁶ Notably, we identified the same missense variant as previously reported, p.(Gly92Ala), in an unrelated individual (22).

According to segregation analysis, minor allele frequency, and the predictive in silico analysis, variants were classified as follows: 23 pathogenic and 10 likely pathogenic.
## Genotype-phenotype correlations

Statistical analysis results are shown in detail in the *Supplemental Analysis*.

A statistically significant correlation was observed regarding the severity of the cerebellar phenotypes when comparing missense variants affecting the DBD with other missense variants and deletions. A *P* value < .05 was observed both in this series of 40 individuals (*P* value = .03365) and in the combined series of 56 individuals from this study and from Guissart et al⁶ affected individuals (*P* value = .02374). It is worth noting that the significance trend correlates with the size of the series.

## Discussion

There is emerging knowledge that the *RORA* gene is of particular importance for cognition and cerebellar function. A first study combining clinical, genetic, and functional experimental data suggested a dual pathological effect of missense variants according to their location in the binding domains.⁶ In this study, we significantly expanded the series of RORA-NDD cases by reporting 40 unpublished individuals with 33 previously undescribed pathogenic and likely pathogenic variants. We also reported the follow-up of 3 previously reported individuals. This allowed us to delve deeper into the phenotype associated with *RORA* variants.

In line with the first study, we confirm that more than half of the RORA-NDD cases are caused by frameshift variants. Almost all missense variants affect a functional domain, 7 out of 9 being located within or slightly upstream of the DBD (Figure 4) and are statistically associated with a more severe cerebellar phenotype. On the other hand, marked cerebellar phenotypes were also observed in individuals with nonsense or frameshift variants, and 9 variants were inherited from an unaffected parent, showing incomplete penetrance.

The missense variant previously reported, p.(Gly92Ala), was also detected in the present series. The 65-year-old individual heterozygous for a pathogenic variant (individual 22) showed a cerebellar syndrome with slowly progressive ataxia and cerebellar and marked upper vermian atrophy. He was not related to the first affected individual with the variant p.(Gly92Ala) reported by Guissart et al⁶ (case 2), for whom we provide follow-up MRI data. The analysis of these images showed that severe progressive cerebellar atrophy could occur in addition to congenital hypoplasia. Furthermore, we observed that the 2 missense variants located in the LBD (p.(Lys339Thr) and p.(Val379Gly)) were associated with a less severe cerebellar phenotype. Statistical correlation between missense variants in DBD and more severe cerebellar signs evidences a significant genotype-phenotype correlation.

Reanalysis of MRIs, when available, revealed cerebellar hypoplasia/atrophy or both in at least half of the individuals. These data indicate that MRI should be done systematically

in individuals with RORA-NDD, with special attention given to the cerebellum (anatomy and biometrics). Our results align with the anatomical expression of *RORA* and with the experimental work reported in literature. Ataxia and severe cerebellar atrophy have been well assessed in the natural strain of the Staggerer mice and in *RORA* null mouse models.³,⁵,⁶ Our clinical observations mirror these experimental findings, indicating early-onset cerebellar signs in the first months of life or since walking milestones are attained. By contrast, potentially disabling *RORA*-associated cerebellar disorders progressed with aging in 5 individuals, and adult-onset cerebellar signs occurred in 6 individuals. Interestingly, in the heterozygous Staggerer (*Rora⁺/sg*) model, an accelerated loss of PC and granule cells was reported to appear with aging. This observation, along with our clinical findings, suggests that RORA may provide neuroprotection beyond typical maturation and remains essential in adulthood for synaptic remodeling and maintaining efficient cerebellar networks.²,¹⁹,²⁰

This study found epilepsy in almost half of RORA-NDD individuals (18/38; 47.4%), which is somewhat less than in the first study (11/16; 69%). The core group presented GGE of variable severity, ranging from seizure free to drug resistant. Interestingly, heterozygous variants in *RORB*, another gene belonging to the ROR family, have been established as causing GGE with mild to moderate ID.²⁰,²¹ Whereas absence seizures are a hallmark of *RORB*-related disorders, myoclonic seizures are more prevalent in RORA-NDD (61%), and the most frequent syndromic diagnosis was EEM or EEM-like syndromes. Both conditions include a subset of individuals with DEE, characterized by multidrug-resistant seizure types.

Experimental data concerning the role of efferent cerebellar pathways in either promoting or inhibiting seizures yield conflicting results.²²⁻²⁴ Three primary electrophysiological hypotheses are under discussion: (1) loss of PC resulting in the disinhibition of the cerebello-thalamic-cortical pathway; (2) neuronal cell loss in the dentate nuclei leading to dysfunctional cortical projections; and (3) reduction of GABA concentrations or GABAergic synapses in the sensorimotor cortex.²⁴ A recent electrophysiological study using anodal cerebellar transcranial direct current stimulation adds further support to the involvement of the cerebellum in the pathophysiology of cortical myoclonus.²⁵ It would be of interest to further analyze the electrophysiological aspects of *RORA* seizure types as they evolve with age to validate if *RORA* is definitely an epilepsy-with-myoclonic-absence-causing gene.

ASD is much less frequent (7/34; 20.6%) in this series than in the previous one (5/12; 42%). Nevertheless, it remains a feature of RORA-NDD. Indeed, intact cerebellocortical connectivity not only supports motor functions but also plays a role in higher cognitive and emotional processes.²⁶ Alterations in these subcircuits could contribute to the cognitive and behavioral phenotype of *RORA*-related disorders. The behavioral problems found in 32.5% of our series could be representative of this contribution. The
pathophysiological contribution of the cerebellum in ASD is increasingly recognized.²⁷,²⁸ RORα1, mainly expressed in PCs, has been validated as a transcriptional regulator of at least 6 ASD-associated genes: A2BP1 (HGNC:18222), CYP19A1 (HGNC:2594), HSD17B10 (HGNC:4800), ITPR1 (HGNC:6180), NLGN1 (HGNC:14291), and NTRK2 (HGNC:8032).²⁹,³⁰ Moreover, as a sex-sensitive gene, RORA was suggested to explain the male predominance in ASD.²⁹ Pharmacological trials related to RORA remain rare, in contrast to the extensive research on therapeutic effects with innate or synthetic agonists of RORα and RORγ in metabolic diseases, immunity, or inflammation.³¹⁻³⁴ Despite this, a recent study in a vitamin-A-deficient mouse showed a significant improvement in motor coordination after vitamin A supplementation, attributed to the upregulation of RORA.³⁵

RORA has also been identified as a circadian gene.³⁶,³⁷ Although this study did not specifically focus on assessing sleep-wake rhythm disorders, anamnestic sleep abnormalities were reported in 33% of our series. The investigation of sleep disturbance should be a priority in future research involving these individuals, potentially leading to therapeutic interventions, such as melatonin-targeted treatment for RORA-related sleep problems.³⁸

Finally, we report a unique case of a RORA in-frame deletion associated with retinal degeneration diagnosed at the age of 21, when vision began to deteriorate. A retinopathy gene panel consisting of 230 genes and an exome sequencing analysis did not show any additional pathogenic variants. RORA is, together with RORB, critically involved in photoreceptor maturation.² Moreover, RORA has been shown to be a key regulator of several homeostatic pathways of the retina and is linked to multiple genes involved in age-related macular degeneration. A recent gene therapeutic study, using AAV5-hRORA as a modifier gene in the Abca4⁻¹⁻² knockout mouse model for macular degeneration, showed promising efficacy; thus, retinal function and molecular outcomes were significantly improved in animals treated earlier or in the intermediate stage of the disease.³⁹

Additional fundus evaluations in individuals with RORA-NDD are important to confirm the causative role of RORA in photoreceptor dysfunction in humans as rescue gene therapies could be available in the future.

## Conclusion

RORA-NDD is characterized by 3 cardinal features: (1) developmental disability, (2) a striking cerebellar phenotype with cerebellar hypoplasia/atrophy or both, often predominantly in the vermis, and (3) epilepsy consisting of a variety of myoclonic seizures. A more severe RORA-associated cerebellar manifestation is caused by missense variants specifically located in the DBD. The age of onset is highly variable, ranging from early infancy to the 5th decade, with progression in a subset of individuals. A careful neurological follow-up is essential, and repeated imaging should be considered when

clinical cerebellar signs occur or progress. We propose RORA as an EEM gene; to corroborate this hypothesis, future detailed analyses of the electrophysiological aspects of seizure types and their evolution with age are needed. A deeper understanding of RORA dysfunction could pave the way to new therapeutic approaches targeting movement control, cognitive, behavioral, sleep issues, and retinal function.

## Data Availability

Data and materials can be supplied upon request to the corresponding author.

## Ethics Declarations

This study was approved by the local institutional Ethics Committee (Comission Nationale de l'Informatique et des Libertés Register Number: 22_5867) and adheres to the principles set out in the Declaration of Helsinki. All data collected were deidentified and handled in accordance with the “Directive on the protection of individuals with regard to the processing of personal data” and the national legislation applicable to it, as required by “The European General Data Protection Regulation” (General Data Protection Regulation [AVG/GDPR]-EU2016/679) and the French legislation elaborating on this regulation. Informed consent was required and obtained from all participants.

## Funding

K.Õ. is supported by Estonian Research Council grants PRG471 and PRG2040.

## Author Contributions

Conceptualization: M.T., G.L.M., J.d.B., S.K.; Data Curation: M.T., J.d.B., G.L.M., M.D.W., S.K., S.C.; Clinical Investigation: J.d.B., M.D.W., X.L.G., G.L.M., M.E., D.B., B.I., B.C., R.P., M.F., L.P., I.G., S.E.H., P.J.B., A.S., C.K., O.V., E.H.G., S.L., I.R., I.K., M.B., K.V., R.C.C., C.S., R.J.T., T.S., S.M., A.N., T.B.H., N.D., H.H., J.J., S.C., G.Y., M.T., G.B., C.G., M.R., A.B., S.R., K.U., A.W., D.T., D.H.K., N.C., S.K., S.M., K.Õ.; Data Analysis: M.T., J.d.B., G.L.M., M.D.W., S.K., S.C.; Writing-original draft: M.T., J.d.B., G.L.M., M.D.W., S.K., S.C.; Writing-review and editing: all co-authors.

## Conflict of Interest

The authors declare no conflicts of interest.
## Additional Information

The online version of this article (https://doi.org/10.1016/j.gim.2024.101347) contains supplemental material, which is available to authorized users.

## Affiliations

¹Department of Medical and Surgical Sciences, University Magna Graecia of Catanzaro, Catanzaro, Italy; ²Genetics Department, Hospices Civils de Lyon, Member of the ERN EpiCARE, Lyon, France; ³Neuromyogene Institute, Pathology and Genetics of neuron and muscle, CNRS UMR 5261 INSERM U1315, University of Lyon-Université Claude Bernard Lyon 1, Lyon, France; ⁴Department of Clinical Epileptology, Sleep Disorders and Functional Neurology in Children, University Hospital of Lyon (HCL), Member of the ERN EpiCARE, Lyon, France; ⁵Department of Pediatric Neurology, Antwerp University Hospital, University of Antwerp, - Edegem, Belgium; ⁶Poitiers University Hospital, Medical Genetics Department, Poitiers, France; ⁷Poitiers University, INSERM U1084, LNEC, Poitiers, France; ⁸Nantes University, ophthalmology department Nantes University Hospital, Inserm, TARGET, Nantes, France; ⁹Medical Genetics Department, Nantes University, CHU Nantes, Nantes, France; ¹⁰The Thorax Institute, INSERM, CNRS, Nantes University, CHU Nantes, Nantes, France; ¹¹Clinical Genetics Department, “Intellectual Deficiencies of Rare Causes” Reference Center (CRDI), CLAD-Ouest Developmental Abnormalities Reference Center, CHU Rennes, Rennes, France; ¹²Centre for Applied Neurogenetics, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada; ¹³Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH; ¹⁴Joe DiMaggio Children's Hospital, Hollywood, FL; ¹⁵Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH; ¹⁶Center for Medical Genetics, Ghent University Hospital, Belgium; ¹⁷Clinical Genetics, University Medical Center Groningen, Groningen, The Netherlands; ¹⁸Center for Neurogenetics, Weill Cornell Medicine, Brain and Mind Research Institute, New York, NY; ¹⁹Department of Medical Genetics, Kazan State University, Russia; ²⁰GENOMED medical-genetic center, Moscow, Russia; ²¹Department of Neurology, Svt. Luka's Institute of Child Neurology and Epilepsy, Moscow, Russia; ²²Institute of Human Genetics, School of Medicine and Health, Technical University of Munich, Munich, German; ²³Department of Obstetrics and Gynecology, Klinikum Rechts der Isar, Technical University of Munich, Munich, Germany; ²⁴Department of Pediatric Neurology and Developmental Medicine and LMU Center for Children with Medical Complexity, Children's Hospital, LMU Hospital, Ludwig-Maximilians-University, Munich, Germany; ²⁵Greenwood Genetic Center, Greenwood, South Carolina;

²⁶Division of Medical Genetics and Genomic Medicine, Vanderbilt University Medical Center, Nashville, TN; ²⁷Pediatric Neurology, Karolinska University Hospital, Stockholm, Sweden; ²⁸Department of Neurology, Charity University Medicine Berlin, Berlin, Germany; ²⁹Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany; ³⁰Center for Neurology and Hertie-Institute for Clinical Brain Research, Tübingen; ³¹Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada; ³²Boston Children's Hospital, department of neurology, Boston Children's Hospital, Boston, MA; ³³Federation of Genetics, Molecular Genetics Department, Necker-Enfants Malades Hospital, Paris, France; ³⁴Neuropediatrics, University Children's Hospital Tübingen, Tübingen, Germany; ³⁵Medical Genetics Department, Brest University Hospital, Brest, France; ³⁶Reference Center for Intellectual Disabilities, Pediatric Department, Brest University Hospital, Brest, France; ³⁷University of Brest, Inserm, EFS, UMR 1078, GGB, Brest, France; ³⁸Department of Neuropediatrics, Jena University Hospital, Jena, Germany; ³⁹Institute of Human Genetics, Jena University Hospital, Jena, Germany; ⁴⁰Department of Neurology and Center for Translational Neuro- and Behavioral Sciences (CTNBS), Essen University Hospital, University of Duisburg-Essen, Essen, Germany; ⁴¹Division of Medical Genetics, Department of Pediatrics, Duke University Medical Center, Durham, NC; ⁴²Department of Clinical Genetics, Genetics and Personalized Medicine Clinic, Tartu University Hospital, Tartu, Estonia; ⁴³Institute of Clinical Medicine, University of Tartu, Tartu, Estonia; ⁴⁴Pediatric imaging department, Femme-Mere-Enfant hospital, Lyon 1 University, Lyon, France

## References

1. Carlberg C, Hooft van Huijsduijnen R, Staple JK, DeLamarter JF, Becker-André M. RZRs, a new family of retinoid-related orphan receptors that function as both monomers and homodimers. *Mol Endocrinol*. 1994;8(6):757-770. http://doi.org/10.1210/mend.8.6.7935491

2. Jetten AM. Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism. *Nucl Recept Signal*. 2009;7:e003. http://doi.org/10.1621/nrs.07003

3. Lee JM, Kim H, Baek SH. Unraveling the physiological roles of retinoic acid receptor-related orphan receptor α. *Exp Mol Med*. 2021;53(9):1278-1286. http://doi.org/10.1038/s12276-021-00679-8

4. Giguère V, Tini M, Flock G, Ong E, Evans RM, Otulakowski G. Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR alpha, a novel family of orphan hormone nuclear receptors. *Genes Dev*. 1994;8(5):538-553. http://doi.org/10.1101/gad.8.5.538

5. Iizuka A, Matsuzaki Y, Konno A, Hirai H. Plasticity of the developmentally arrested staggerer cerebellum in response to exogenous RORα. *Brain Struct Funct*. 2016;221(6):2879-2889. http://doi.org/10.1007/s00429-015-1077-9

6. Guissart C, Latypova X, Rollier P, et al. Dual molecular effects of dominant RORA mutations cause two variants of syndromic intellectual disability with either autism or cerebellar ataxia. *Am J Hum Genet*. 2018;102(5):744-759. http://doi.org/10.1016/j.ajhg.2018.02.021
7. Haerian BS, Sha'ari HM, Tan HJ, et al. RORA gene rs12912233 and rs880626 polymorphisms and their interaction with SCN1A rs3812718 in the risk of epilepsy: a case-control study in Malaysia. *Genomics*. 2015;105(4):229-236. http://doi.org/10.1016/j.ygeno.2015.02.001

8. Hu VW. Is retinoic acid-related orphan receptor-alpha (RORA) a target for gene-environment interactions contributing to autism? *Neurotoxicology*. 2012;33(6):1434-1435. http://doi.org/10.1016/j.neuro.2012.07.009

9. Arnaud L, Abi Warde MT, Barcia G, et al. The EPIGENE network: a French initiative to harmonize and improve the nationwide diagnosis of monogenic epilepsies. *Eur J Med Genet*. 2022;65(3):104445. http://doi.org/10.1016/j.ejmg.2022.104445

10. Zuberi SM, Wirrell E, Yozawitz E, et al. ILAE classification and definition of epilepsy syndromes with onset in neonates and infants: position statement by the ILAE Task Force on Nosology and Definitions. *Epilepsia*. 2022;63(6):1349-1397. http://doi.org/10.1111/epi.17239

11. American Psychiatric Association. In: *Diagnostic and Statistical Manual of Mental Disorders: DSM-5*. 5th ed. American Psychiatric Association; 2013. http://doi.org/10.1176/appi books.9780890425596

12. Freeman PJ, Hart RK, Gretton LJ, Brookes AJ, Dalgleish R. VariantValidator: accurate validation, mapping, and formatting of sequence variation descriptions. *Hum Mutat*. 2018;39(1):61-68. http://doi.org/10.1002/humu.23348

13. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. *Nat Protoc*. 2009;4(7):1073-1081. http://doi.org/10.1038/nprot.2009.86

14. Rentzsch P, Witten D, Cooper GM, Shendure J, Kircher M. CADD: predicting the deleteriousness of variants throughout the human genome. *Nucleic Acids Res*. 2019;47(D1):D886-D894. http://doi.org/10.1093/nar/gky1016

15. Ioannidis NM, Rothstein JH, Pejaver V, et al. REVEL: an ensemble method for predicting the pathogenicity of rare missense variants. *Am J Hum Genet*. 2016;99(4):877-885. http://doi.org/10.1016/j.ajhg.2016.08.016

16. Wiel L, Baakman C, Gilissen D, Veltman JA, Vriend G, Gilissen C. MetaDome: pathogenicity analysis of genetic variants through aggregation of homologous human protein domains. *Hum Mutat*. 2019;40(8):1030-1038. http://doi.org/10.1002/humu.23798

17. Tordai H, Torres O, Csepi M, Padányi R, Lukács GL, Hegedűs T. Analysis of AlphaMissense data in different protein groups and structural context. *Sci Data*. 2024;11:495. http://doi.org/10.1038/s41597-024-03327-8

18. Nykamp K, Anderson M, Powers M, et al. Sherloc: a comprehensive refinement of the ACMG-AMP variant classification criteria. *Genet Med*. 2017;19(10):1105-1117. http://doi.org/10.1038/gim.2017.37 [published correction appears in *Genet Med*. 2020;22(1):240. doi:10.1038/s41436-019-0624-9].

19. Janmaat S, Akwa Y, Doulazmi M, et al. Age-related Purkinje cell death is steroid dependent: RORα haplo-insufficiency impairs plasma and cerebellar steroids and Purkinje cell survival. *Age (Dordr)*. 2011;33(4):565-578. http://doi.org/10.1007/s11357-010-9203-3

20. Vitalis T, Mariani J. Retinoid receptor-related orphan receptor alpha: a key gene setting brain circuits. *Neural Regen Res*. 2018;13(5):791-794. http://doi.org/10.4103/1673-5374.232462

21. Gokce-Samar Z, Vetro A, De Bellescize J, et al. Molecular and phenotypic characterization of the RORB-related disorder. *Neurology*. 2024;102(2):e207945. http://doi.org/10.1212/WNL.0000000000207945

22. Streng ML, Krook-Magnuson E. The cerebellum and epilepsy. *Epilepsy Behav*. 2021;121(B):106909. http://doi.org/10.1016/j.yebeh.2020.106909

23. Marcián V, Filip P, Bareš M, Brázdil M. Cerebellar dysfunction and ataxia in patients with epilepsy: coincidence, consequence, or cause? [published correction appears in Tremor Other Hyperkinet Mov (N Y). 2016;6:416. doi:10.7916/D80Z73DZ]. http://doi.org/10.7916/D8KH0NBT

24. van der Veen S, Zutt R, Klein C, et al. Nomenclature of genetically determined myoclonus syndromes: recommendations of the international Parkinson and Movement Disorder Society task force. *Mov Disord*. 2019;34(11):1602-1613. http://doi.org/10.1002/mds.27828

25. Latorre A, Rocchi L, Paparella G, Manzo N, Bhatia KP, Rothwell JC. Changes in cerebellar output abnormally modulates cortical myoclonus sensorimotor hyperexcitability. *Brain*. 2024;147(4):1412-1422. http://doi.org/10.1093/brain/awad384

26. Schmahmann JD. The cerebellum and cognition. *Neurosci Lett*. 2019;688:62-75. http://doi.org/10.1016/j.neulet.2018.07.005

27. Fatemi SH, Reutiman TJ, Folsom TD, Sidwell RW. The role of cerebellar genes in pathology of autism and schizophrenia. *Cerebellum*. 2008;7(3):279-294. http://doi.org/10.1007/s12311-008-0017-0

28. Wang SSH, Kloth AD, Badura A. The cerebellum, sensitive periods, and autism. *Neuron*. 2014;83(3):518-532. http://doi.org/10.1016/j.neuron.2014.07.016

29. Sarachana T, Hu VW. Genome-wide identification of transcriptional targets of RORA reveals direct regulation of multiple genes associated with autism spectrum disorder. *Mol Autism*. 2013;4(1):14. http://doi.org/10.1186/2040-2392-4-14

30. Leblond CS, Le TL, Malesys S, et al. Operative list of genes associated with autism and neurodevelopmental disorders based on database review. *Mol Cell Neurosci*. 2021;113:103623. http://doi.org/10.1016/j.mcn.2021.103623

31. Chai C, Cox B, Yaish D, et al. Agonist of RORA attenuates nonalcoholic fatty liver progression in mice via up-regulation of microRNA 122. *Gastroenterology*. 2020;159(3):999-1014.e9. http://doi.org/10.1053/j.gastro.2020.05.056

32. Kojetin DJ, Burris TP. REV-ERB and ROR nuclear receptors as drug targets. *Nat Rev Drug Discov*. 2014;13(3):197-216. http://doi.org/10.1038/nrd4100

33. Matta-Camacho E, Banerjee S, Hughes TS, et al. Structure of REV-ERBβ ligand-binding domain bound to a porphyrin antagonist. *J Biol Chem*. 2014;289(29):20054-20066. http://doi.org/10.1074/jbc.M113.545111

34. Brożyna AA, Żmijewski MA, Linowiecka K, Kim TK, Slominski RM, Slominski AT. Disturbed expression of vitamin D and retinoic acid-related orphan receptors α and γ and of megalin in inflammatory skin diseases. *Exp Dermatol*. 2022;31(5):781-788. http://doi.org/10.1111/exd.14521

35. Wang S, Liu H, Cheng B, et al. Vitamin A supplementation ameliorates motor incoordination via modulating RORα in the cerebellum in a valproic acid-treated rat autism model with vitamin A deficiency. *Neurotoxicology*. 2021;85:90-98. http://doi.org/10.1016/j.neuro.2021.05.004

36. Zhang J, Zhao L, Li Y, et al. Circadian clock regulates granulosa cell autophagy through NR1D1-mediated inhibition of ATG5. *Am J Physiol Cell Physiol*. 2022;322(2):C231-C245. http://doi.org/10.1152/ajpcell.00267.2021

37. Sato TK, Panda S, Miraglia LJ, et al. A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. *Neuron*. 2004;43(4):527-537. http://doi.org/10.1016/j.neuron.2004.07.018

38. Tosini G, Davidson AJ, Fukuhara C, Kasamatsu M, Castanon-Cervantes O. Localization of a circadian clock in mammalian photo-receptors [published correction appears in FASEB J. 2008 Mar;22(3):941]. *FASEB J*. 2007;21(14):3866-3871. http://doi.org/10.1096/fj.07-8371com

39. Akula M, McNamee SM, Love Z, et al. Retinoic acid related orphan receptor α is a genetic modifier that rescues retinal degeneration in a mouse model of Stargardt disease and Dry AMD. *Gene Ther*. 2024;31(7-8):413-421. http://doi.org/10.1038/s41434-024-00455-z