# Clinical and genetic delineation of autosomal recessive and dominant ACTL6B-related developmental brain disorders

Elisa Cali, Tania Quirin, Clarissa Rocca, Stephanie Efthymiou, Antonella Riva, Dana Marafi, Maha Zaki, Mohnish Suri, Roberto Dominguez, Hasnaa Elbendary, et al.

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## To cite this version:

Elisa Cali, Tania Quirin, Clarissa Rocca, Stephanie Efthymiou, Antonella Riva, et al. Clinical and genetic delineation of autosomal recessive and dominant ACTL6B-related developmental brain disorders. Genetics in Medicine, 2024, pp.101251. 10.1016/j.gim.2024.101251. hal-05020584

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ELSEVIER

ARTICLE

Clinical and genetic delineation of autosomal
recessive and dominant ACTL6B-related
developmental brain disorders

Elisa Cali¹ ID, Tania Quirin², Clarissa Rocca¹, Stephanie Efthymiou¹, Antonella Riva³,
Dana Marafi⁴,⁵, Maha S. Zaki⁶, Mohnish Suri⁷,⁸, Roberto Dominguez⁹,
Hasnaa M. Elbendary⁶, Shahryar Alavi¹,¹⁰, Mohamed S. Abdel-Hamid¹¹, Heba Morsy¹,¹²,
Frederic Tran Mau-Them¹³,¹⁴, Mathilde Nizon¹⁵,¹⁶, Pavel Tesner¹⁷, Lukáš Ryba¹⁷,
Faisal Zafar¹⁸, Nuzhat Rana¹⁸, Nebal W. Saadi¹⁹, Zahra Firoozfar¹⁰, Pinar Gencpinar²⁰,
Bulent Unay²¹, Canan Ustun²¹, Ange-Line Bruel²²,²³, Christine Coubes²⁴,
Jennifer Stefanich²⁵, Ozlem Sezer²⁶, Emanuele Agolini²⁷, Antonio Novelli²⁷,
Gessica Vasco²⁸, Donatella Lettori²⁸, Mathieu Milh²⁹, Laurent Villard³⁰, Shimriet Zeidler³¹,
Henry Opperman³², Vincent Strehlow³², Mahmoud Y. Issa⁶, Hebatallah El Khassab³³,
Prem Chand³⁴, Shahnaz Ibrahim³⁴, Ali Rashidi-Nezhad³⁵,³⁶, Mohammad Miryounesi³⁷,
Pegah Larki³⁷, Jennifer Morrison³⁸, Ingrid Cristian³⁸, Isabelle Thiffault³⁹,⁴⁰,⁴¹,
Nicole L. Bertsch⁴², Grace J. Noh⁴³, John Pappas⁴⁴,⁴⁵, Ellen Moran⁴⁶,
Nikolaos M. Marinakis⁴⁷, Joanne Traeger-Synodinos⁴⁷, Susan Hosseini⁴⁸,
Mohammad Reza Abbaszadegan⁴⁹, Roseline Caumes⁵⁰, Lisenka E.L.M. Vissers⁵¹,⁵²,
Maedeh Neshatdoust⁵³, Mostafa Montazer Zohour⁵⁴, Elmostafa El Fahime⁵⁵,
Christina Canavati⁵⁶, Lara Kamal⁵⁶, Moien Kanaan⁵⁶, Omar Askander⁵⁷,
Victoria Voinova⁵⁸,⁵⁹, Olga Levchenko⁶⁰, Shahzhad Haider⁶¹, Sara S. Halbach⁶²,
Rayana Elias Maia⁶³, Salehi Mansoor⁶⁴,⁶⁵, Vivek Jain⁶⁶, Sanjukta Tawde⁶⁷,
Viveka Santhosh R. Challa⁶⁸, Vykuntaraju K. Gowda⁶⁸, Varunvenkat M. Srinivasan⁶⁸,
Lucas Alves Victor⁶⁹, Benito Pinero-Banos⁷⁰, Jennifer Hague⁷¹, Heba Ahmed ElAwady⁷²,
Adelia Maria de Miranda Henriques-Souza⁷³, Huma Arshad Cheema⁷⁴,
Muhammad Nadeem Anjum⁷⁴, Sara Idkaidak⁷⁵, Firas Alqarajeh⁷⁶, Osama Atawneh⁷⁶,
Hagar Mor-Shaked⁷⁷, Tamar Harel⁷⁷, Giovanni Zifarelli⁷⁸, Peter Bauer⁷⁸, Fernando Kok⁷⁹,
Joao Paulo Kitajima⁷⁹, Fabiola Monteiro⁷⁹, Juliana Josahkian⁷⁹, Gaetan Lesca⁸⁰,⁸¹,
Nicolas Chatron⁸⁰,⁸¹, Dorothe Ville⁸², David Murphy⁸³, Jeffrey L. Neul⁸⁴,

The Article Publishing Charge (APC) for this article was paid by Wellcome Trust (WT093205MA and WT104033AIA).

*Correspondence and requests for materials should be addressed to Reza Maroofian, Department of Neuromuscular Diseases, University College London, Queen Square, Institute of Neurology, WC1N 3BG London, UK. Email address: r.maroofian@ucl.ac.uk

Affiliations are at the end of the document.
Sureni V. Mullegama<sup>85</sup>, Amber Begtrup<sup>85</sup>, Isabella Herman<sup>86</sup>, Tadahiro Mitani<sup>4</sup>, Jennifer E. Posey<sup>4</sup>, Chee Geap Tay<sup>87</sup>, Iram Javed<sup>88</sup>, Lucinda Carr<sup>89</sup>, Farah Kanani<sup>90</sup>, Fiona Beecroft<sup>90</sup>, Lee Hane<sup>91</sup>, Elsayed Abdelkreem<sup>92</sup>, Milan Macek<sup>17</sup>, Luciana Bispo<sup>93</sup>, Marwa Abd Elmaksoud<sup>94</sup>, Farzad Hashemi-Gorji<sup>95</sup>, Davut Pehlivan<sup>4,86</sup>, David J. Amor<sup>96</sup>, Rami Abou Jamra<sup>32</sup>, Wendy K. Chung<sup>97</sup>, Eshan Ghayoor Karimiani<sup>98,99,100</sup>, Philippe M. Campeau<sup>101,102</sup>, Fowzan S. Alkuraya<sup>103</sup>, Alistair T. Pagnamenta<sup>104</sup>, Joseph G. Gleeson<sup>105,106</sup>, James R. Lupski<sup>4,107,108</sup>, Pasquale Striano<sup>3,109</sup>, Andres Moreno-De-Luca<sup>110</sup>, Denis L.J. Lafontaine<sup>2 ID</sup>, Henry Houlden<sup>1</sup>, Reza Maroofian<sup>1,* ID</sup>

## ARTICLE INFO

### Article history:

Received 7 December 2023

Received in revised form

4 September 2024

Accepted 5 September 2024

Available online 17 September 2024

### Keywords:

ACTL6B

Autism

BAFopathies

Epileptic-dyskinetic encephalopathy

Ribosomopathies

## ABSTRACT

**Purpose:** This study aims to comprehensively delineate the phenotypic spectrum of ACTL6B-related disorders, previously associated with both autosomal recessive and autosomal dominant neurodevelopmental disorders. Molecularly, the role of the nucleolar protein ACTL6B in contributing to the disease has remained unclear.

**Methods:** We identified 105 affected individuals, including 39 previously reported cases, and systematically analyzed detailed clinical and genetic data for all individuals. Additionally, we conducted knockdown experiments in neuronal cells to investigate the role of ACTL6B in ribosome biogenesis.

**Results:** Biallelic variants in ACTL6B are associated with severe-to-profound global developmental delay/intellectual disability, infantile intractable seizures, absent speech, autistic features, dystonia, and increased lethality. De novo monoallelic variants result in moderate-to-severe global developmental delay/intellectual disability, absent speech, and autistic features, whereas seizures and dystonia were less frequently observed. Dysmorphic facial features and brain abnormalities, including hypoplastic corpus callosum, and parenchymal volume loss/atrophy, are common findings in both groups. We reveal that in the nucleolus, ACTL6B plays a crucial role in ribosome biogenesis, particularly in pre-rRNA processing.

**Conclusion:** This study provides a comprehensive characterization of the clinical spectrum of both autosomal recessive and dominant forms of ACTL6B-associated disorders. It offers a comparative analysis of their respective phenotypes provides a plausible molecular explanation and suggests their inclusion within the expanding category of "ribosomopathies."

© 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

Actin-like 6B (ACTL6B, HGNC:160) encodes an actin-related protein that constitutes part of the neuronal BRG1/brm-associated factor (nBAF) complex.¹ The BAF complex, also known as SWI/SNF complex, comprises at least 15 subunits that induce conformational changes in nucleosomes, thereby regulating chromatin structure and DNA accessibility in an ATP-dependent manner.²,³ The nBAF complex plays a key role in epigenetic regulatory mechanisms guiding neurodevelopmental processes, including neurogenesis (cell proliferation and differentiation), cell migration, maturation, and neuronal integration.⁴

ACTL6B (also known as BAF53B) is specifically found in nBAF complexes of post-mitotic neurons where it appears to play a critical role in activity-dependent dendritic outgrowth and neuronal maturation.⁵ Deficits in distinct subunits of this

complex have been linked to a range of syndromic or non-syndromic neurodevelopmental disorders (NDD). Pathogenic loss-of-function (LoF) or dominant negative (DN)/gain-of-function (GoF) variants in numerous subunits, such as ARID1A (HGNC:11110), ARID1B (HGNC:18040), ARID2 (HGNC:18037), SMARCC2 (HGNC:11105), SMARCD1 (HGNC:11106) (LoF) and SMARCA4 (HGNC:11100), SMARCE1 (HGNC:11109), SMARCB1 (HGNC:11103)(DN/GoF), cause Coffin-Siris syndrome, which is an autosomal dominant disorder characterized by intellectual disability/ global developmental delay (ID/GDD), speech impairment, feeding difficulties, coarse facial features, hypertrichosis, hypoplastic fifth finger and toenails, and corpus callosum agenesis⁶⁻⁸ (MIM 135900, 614607, 614608, 614609, 616938, 617808, 618027, 618362, and 618779). Furthermore, pathogenic heterozygous variants in SMARCA2 (HGNC:11098) are associated with 2 different conditions: Nicolaides-Baraitser
syndrome, characterized by severe GDD/ID, early-onset seizures, short stature, dysmorphic facial features, and sparse hair (MIM 601358), and a congenital disorder that comprises distinct facial features, blepharophimosis, and GDD (blepharophimosis-intellectual impairment syndrome, MIM 619293).⁹,¹⁰ Pathogenic variants in *BCL11A* are associated with an autosomal dominant intellectual developmental disorder with dysmorphic features and asymptomatic persistence of fetal hemoglobin, referred to as Dias Logan syndrome (MIM 617101),¹¹ whereas monoallelic variants in *BCL11B* are linked to either intellectual developmental disorder with dysmorphic facies, speech delay, and T cell abnormalities (MIM 618092) or severe combined immunodeficiency with intellectual disability, spasticity, and craniofacial abnormalities (MIM 617237).¹² Biallelic pathogenic variants in *ACTL6B* have been associated with a subtype of developmental and epileptic encephalopathy (DEE) characterized by severe GDD, epilepsy, cerebral atrophy, and abnormal central system myelination (DEE76, MIM 618468).⁵,¹³⁻¹⁷ Additionally, 2 recurrent de novo missense *ACTL6B* variants have been linked with a dominant NDD characterized by intellectual disability, and severe speech and gait disturbances (intellectual developmental disorder with severe speech and ambulation defects, MIM 618470).⁵ Since 2015, 39 patients from 31 unrelated families carrying either biallelic (29 patients) or de novo (10 patients) variants have been reported in 6 separate publications.⁵,¹³⁻¹⁷ Nevertheless, a comprehensive study describing the full range of genotypic and phenotypic manifestations associated with *ACTL6B* variants is lacking.

In a high-throughput study, ACTL6B was tentatively assigned to the nucleolus,¹⁸ a multi-layered biomolecular condensate in which the initial steps of ribosome biogenesis occur. Ribosome biogenesis disorders, collectively referred to as “ribosomopathies,” encompass an emerging class of syndromes, including neurological disorders, caused by pathogenic variants in ribosomal proteins or in ribosomal assembly factors.¹⁹ Despite partial elucidation of the nBAF complex and ACTL6B protein's role in neuronal development, especially in association with DEE or NDD, no study has suggested ACTL6B's involvement in ribosome biogenesis so far. In this study, we define the spectra of *ACTL6B*-related disorders in 105 patients from 90 unrelated families, including 65 newly described patients harboring either biallelic (n = 50) or de novo (n = 16) variants in *ACTL6B*. In addition, we report a significant role of ACTL6B in ribosome biogenesis, providing a likely molecular explanation for the associated disorders. Thus, we propose that *ACTL6B*-related disorders are new forms of “ribosomopathies.”

# Materials and Methods

## Patient ascertainment: Clinical and genetic evaluation

The affected individuals were identified through extensive data sharing with collaborators and database screening of several

diagnostic and research genetic laboratories worldwide, as well as using GeneMatcher.²⁰ Informed consents were obtained from all study participants and/or their parents or legal guardians. Genome/exome sequencing was performed on genomic DNA extracted from blood in different diagnostic or research laboratories worldwide. When necessary, candidate variants were confirmed via Sanger sequencing in additional family members, especially in cases of presumed de novo variants or informative families with multiple affected or unaffected siblings. Comprehensive clinical data, including epileptic phenotype details (seizure duration/frequency) and family histories, were systematically collected from new and reported cases by recruiting clinicians and documented in structured data forms. Where available, electroencephalogram (EEG) recordings, clinical photos, videos, and brain magnetic resonance imaging (MRI) scans were reviewed by a multidisciplinary team comprising child neurologists, epileptologists, dysmorphologists, and neuroradiologists.

## In silico analysis

Information on the ACTL6B protein was obtained from the AlphaFold Protein Structure database.²¹ Protein tertiary structures were visualized using PyMOL (Schrödinger, LLC) and the variants were modeled in Coot.²² All variants were standardized to NM_016188.5 /GRCh38/hg38.

## Cellular studies

Cells were cultured under standard conditions. Knockdown experiments were carried out by reverse transfecting SH-SY5Y cells with dicer-substrate short interfering RNAs (DsiRNA) silencers (integrated DNA technologies), followed by total RNA extraction for subsequent denaturing RNA electrophoresis and northern blot analysis. Western blotting was performed on cell lysates collected 72 hours post-dicer-substrate short interfering RNAs treatment, with proteins separated by SDS-PAGE and transferred to a nitrocellulose membrane for detection using the BAF53B polyclonal antibody. Immunofluorescence assays were performed on SH-SY5Y and U2OS cells using antibodies targeting ACTL6B and nucleolar components. The acquired images were analyzed using FIJI (ImageJ).²³ Detailed explanation of the methods is available in the Supplemental Methods.

# Results

## Clinical characterization of disease spectrum

The comprehensive cohort, encompassing both autosomal recessive and dominant cases, comprises 105 patients from 90 unrelated families, with 62 females and 42 males (Supplementary Tables 1 and 2). Of these, 65 individuals are newly described, with ages at last evaluation ranging
from 3 months to 29 years (median 4, IQR 6.5). Table 1 provides an overview of the key clinical findings.

Autosomal recessive ACTL6B-related developmental and epileptic-dyskinetic encephalopathy

The autosomal recessive cohort comprises 46 females and 32 males from 65 families, with age at last evaluation ranging between 3 months and 17 years (median 4, IQR 7). Consanguinity was reported in 40 families. Compound heterozygous biallelic variants occurred in 6 of the 16 families without a historical report of consanguinity. Pregnancy and delivery were unremarkable for 26 of the 34 patients with available information. Prenatal manifestations, such as fetal abnormal or slow movements, oligohydramnios, and breech presentation, occurred in 2 fetuses requiring cesarean delivery. Preterm birth was reported in 8 infants, whereas 41 infants were born at term and 1 post-term (median 39 weeks, IQR 1.75). Birth parameters, including length and weight, when available, were within normal ranges for 85% and 89% of the newborns, respectively, whereas head circumference was reduced only in 1 case. Infantile hypotonia manifested in 34 infants (34/45, 76%). Failure to thrive (FTT) was documented in 95% of the children (53/56) and was likely due to feeding difficulties, often detected at birth or in infancy (42/46, 91%) and requiring the use of nasogastric tube or gastrostomy (n = 17).

Seizures manifested in 93% of the children (70/75) with the onset ranging from birth to 11 years (median age 2 months, IQR 6). A majority (81%) experienced their first seizure within the first year of life and 34% within the first month. In 4 individuals (6%), seizures occurred at birth and probably antenatally as well. Patients presented with both focal (n = 18) and generalized (n = 40) onset of seizures. Two exhibited focal-onset seizures with subsequent generalization, whereas 5 experienced only focal-onset seizures, and 26 exclusively had generalized seizures. Seizure types were variable and included myoclonic (n = 30, 8 focal, 17 generalized, 5 not specified), tonic (n = 27, 8 focal, 17 generalized, 2 not specified), tonic-clonic (n = 11, 2 focal with secondary generalization, 10 generalized), atonic (n = 3), and epileptic spasms (n = 12, 2 generalized, 10 not specified). Seizure type and onset were not specified for 16 and 13 individuals, respectively. When data on frequency were available, 92% of the patients presented seizures daily (up to 20-25/day), and clustering was reported in 64% of the cases (20/31). Seizure duration ranged from a few seconds to 5 minutes, and status epilepticus was reported in 10 children (12/32, 38%). When information on treatment was present, seizures were considered intractable in 88% of the patients. Treatment often included a combination of 2 to 5 antiseizure medications (ASMs) because seizures were rarely controlled by a single ASM (4/34, 12%). Although a distinct treatment pattern could not be determined, effective approaches often involved the combination of sodium valproate and other ASMs. Detailed information on the epileptic phenotype and individual treatment is available in Supplemental Table 3.

EEG abnormalities were recorded in 96% of the patients (53/55), including focal, multifocal, and diffuse epileptiform discharges, slowing of background activity, and subcortical changes. Notably, 1 patient exhibited EEG abnormalities despite the absence of clinical seizures. Although there were no distinctive or shared patterns among the abnormalities, certain age-related characteristics were found. A comprehensive summary of the results can be found in Supplemental Materials.

GDD and ID were reported in all the affected individuals (79/79, 100%). Motor delay was observed in 95% of individuals (62/65), with 74% failing to achieve unsupported sitting and 82% unable to accomplish walking (Figure 1D). A significant proportion (61/70, 87%) showed a complete lack of speech and language development, whereas the other 8 individuals (8/70, 11%) exhibited delayed speech, limited just to a few words. Ten patients had regression of motor and cognitive abilities (10/46, 22%). When formal cognitive assessments were performed, the degree of ID varied from moderate (n = 3), to severe (n = 29) and profound (n = 22). Behavioral abnormalities included attention deficit hyperactivity disorder, autism spectrum disorder (ASD), aggressivity, and sleep disturbance. Autistic-like features, such as sound hypersensitivity and stereotypic hand movements, were noted in 26 patients (26/42, 62%).

At the last evaluation, 78% of the patients (45/58) exhibited microcephaly (z-score range -7.5 to +1.5 SD). Notably, head circumference within normal ranges at birth suggested postnatal deceleration of head growth, indicative of progressive microcephaly. Tragically, 23% of patients succumbed to the disorder in infancy or early childhood (median age 2.83 years). The neurological examination showed limb hypertonia and spasticity in a substantial percentage of cases (53/70, 76%). Dystonia was observed in 55% of individuals (32/58), accompanied by increased deep tendon reflexes (35/52, 67%), muscle weakness (33/43, 77%), and axial hypotonia (34/70, 49%). The ophthalmological evaluation revealed a variable presence of refractive defects (n = 9), strabismus (n = 3), and nystagmus (n = 4) (16/31, 52%).

Neuroimaging studies were conducted on 63 individuals. Among these, 28 underwent independent review, whereas radiology reports were available for 35 (Supplementary Table 4). Of those with imaging available for independent review, most cases had low-resolution snapshots of brain MRIs and 3 had DICOM files. Fifty-two individuals (83%) exhibited abnormal findings, whereas 11 reportedly had a normal study (not independently reviewed). Of the reviewed cases (n = 28), the most common abnormalities included hypoplastic corpus callosum (96%), widened subarachnoid spaces (89%), global brain atrophy (86%), ventricular enlargement/ventriculomegaly (57%), white matter volume loss (57%), mega cisterna magna/enlarged cisterna magna (50%), hypomyelination (43%), and patchy white matter signal abnormalities/focal white matter lesions (21%).

Facial photographs and/or videos were reviewed for 29 children from 23 families (Figure 2A). The predominant
<table><caption>**Table 1** Overview of ACTL6B-related clinical features</caption><thead><tr><th rowspan="2">Features</th><th rowspan="2">HPO Codes</th><th colspan="3">Recessive Cases</th><th colspan="3">Dominant Cases</th></tr><tr><th>Newly Reported</th><th>Previously Reported</th><th>Total</th><th>Newly Reported</th><th>Previously Reported</th><th>Total</th></tr></thead><tbody><tr><td>Gender</td><td>-</td><td>29 F, 20M</td><td>17F, 12M</td><td>46F, 32M</td><td>8F, 8M</td><td>8F, 2M</td><td>16F, 10M</td></tr><tr><td>Median age at last follow-up (IQR)</td><td>-</td><td>4.875 (7.25)</td><td>2.25 (3.75)</td><td>4 (7)</td><td>6 (2.87)</td><td>5.7 (4)</td><td>6 (9)</td></tr><tr><td>Zygosity</td><td>-</td><td>42 H, 8 CH</td><td>23H, 6 CH</td><td>65 H, 14 CH</td><td>15DN</td><td>10DN</td><td>25DN</td></tr><tr><td>Growth</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Congenital microcephaly</td><td>HP:0011451</td><td>1/27 (4%)</td><td>- (0/7)</td><td>1/34 (3%)</td><td>2/9 (22%)</td><td>1/2 (50%)</td><td>3/11 (27%)</td></tr><tr><td>Progressive microcephaly</td><td>HP:0000253</td><td>24/34 (71%)</td><td>21/24 (88%)</td><td><strong>45/58 (78%)</strong></td><td>4/12 (33%)</td><td>5/10 (50%)</td><td>9/22 (41%)</td></tr><tr><td>Feeding difficulties</td><td>HP:0011968</td><td>41/45 (91%)</td><td>+ (1/1)</td><td><strong>42/46 (91%)</strong></td><td>8/15 (53%)</td><td>n/a</td><td><strong>8/15 (53%)</strong></td></tr><tr><td>Failure to thrive</td><td>HP:0001508</td><td>40/42 (95%)</td><td>13/14 (93%)</td><td><strong>53/56 (95%)</strong></td><td>5/13 (38%)</td><td>n/a</td><td>5/13 (38%)</td></tr><tr><td>Short stature</td><td>HP:0004322</td><td>16/29 (55%)</td><td>7/12 (58%)</td><td>23/41 (56%)</td><td>- (0/14)</td><td>n/a</td><td>- (0/16)</td></tr><tr><td>Hypotonia in infancy</td><td>HP:0008947</td><td>33/44 (75%)</td><td>+ (1/1)</td><td><strong>34/45 (76%)</strong></td><td>14/16 (88%)</td><td>n/a</td><td><strong>13/15 (87%)</strong></td></tr><tr><td>Motor development</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Motor delay</td><td>HP:0001270</td><td>45/48 (94%)</td><td>17/17 (100%)</td><td><strong>62/65 (95%)</strong></td><td>16/16 (100%)</td><td>8/9 (89%)</td><td><strong>24/25 (96%)</strong></td></tr><tr><td>Unsupported sitting not achieved</td><td>-</td><td>32/45 (71%)</td><td>5/5 (100%)</td><td><strong>37/50 (74%)</strong></td><td>3/10 (30%)</td><td>1/3 (33%)</td><td>4/13 (30%)</td></tr><tr><td>Unsupported walking not achieved</td><td>-</td><td>36/45 (80%)</td><td>5/5 (100%)</td><td><strong>41/50 (82%)</strong></td><td>7/12 (58%)</td><td>4/7 (57%)</td><td>11/19 (61%)</td></tr><tr><td>Intellectual development</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Absent speech</td><td>HP:0001344</td><td>39/48 (81%)</td><td>22/22 (100%)</td><td><strong>61/70 (87%)</strong></td><td>12/16 (75%)</td><td>9/10 (90%)</td><td><strong>21/26 (81%)</strong></td></tr><tr><td>Delayed speech</td><td>HP:0000750</td><td>8/48 (17%)</td><td>-</td><td>8/70 (11%)</td><td>4/16 (25%)</td><td>-</td><td>4/26 (15%)</td></tr><tr><td>GDD/ID</td><td>HP:0001263<br>HP:0001249</td><td>50/50 (100%)</td><td>29/29 (100%)</td><td><strong>79/79 (100%)</strong></td><td>16/16 (100%)</td><td>10/10 (100%)</td><td><strong>26/26 (100%)</strong></td></tr><tr><td>Mild-to-moderate</td><td>-</td><td>2</td><td>1</td><td>3</td><td>4</td><td>-</td><td>4</td></tr><tr><td>Severe</td><td>-</td><td>19</td><td>10</td><td>29</td><td>7</td><td>10</td><td>17</td></tr><tr><td>Profound</td><td>-</td><td>21</td><td>1</td><td>22</td><td>3</td><td>-</td><td>3</td></tr><tr><td>Autistic features</td><td>HP:0000729</td><td>17/32 (53%)</td><td>9/10 (90%)</td><td><strong>26/42 (62%)</strong></td><td>7/13 (54%)</td><td>7/8 (88%)</td><td><strong>14/21 (67%)</strong></td></tr><tr><td>Neurological examination</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Seizures</td><td>HP:0001250</td><td>44/49 (90%)</td><td>26/26 (100%)</td><td><strong>70/75 (93%)</strong></td><td>1/16 (6%)</td><td>1/10 (10%)</td><td>2/26 (8%)</td></tr><tr><td>Increased tendon reflexes</td><td>HP:0001347</td><td>27/40 (68%)</td><td>8/12 (67%)</td><td><strong>35/52 (67%)</strong></td><td>4/9 (44%)</td><td>n/a</td><td>4/9 (44%)</td></tr><tr><td>Hypotonia</td><td>HP:0001252</td><td>24/45 (53%)</td><td>10/25 (40%)</td><td>34/70 (49%)</td><td>9/15 (60%)</td><td>5/6 (83%)</td><td><strong>14/21 (67%)</strong></td></tr><tr><td>Hypertonia/spasticity</td><td>HP:0001276</td><td>31/45 (69%)</td><td>22/25 (88%)</td><td><strong>53/70 (76%)</strong></td><td>5/15 (33%)</td><td>- (0/5)</td><td>5/20 (25%)</td></tr><tr><td>Muscle weakness</td><td>HP:0001324</td><td>31/41 (76%)</td><td>+ (2/2)</td><td><strong>33/43 (77%)</strong></td><td>11/16 (69%)</td><td>n/a</td><td>11/16 (69%)</td></tr><tr><td>Dystonia</td><td>HP:0001332</td><td>19/42 (45%)</td><td>13/16 (81%)</td><td>32/58 (55%)</td><td>4/15 (27%)</td><td>n/a</td><td>4/15 (27%)</td></tr><tr><td>Limb contractures</td><td>HP:0003121</td><td>14/37 (38%)</td><td>- (0/3)</td><td>14/40 (35%)</td><td>1/14 (7%)</td><td>n/a</td><td>1/14 (7%)</td></tr><tr><td>Dyskinetic movements</td><td>HP:0100022</td><td>10/38 (26%)</td><td>2/3 (67%)</td><td>12/41 (29%)</td><td>8/15 (53%)</td><td>n/a</td><td>8/15 (53%)</td></tr><tr><td>Brain MRI abnormalities</td><td>HP:0410263</td><td>28/38 (73%)</td><td>24/25 (96%)</td><td><strong>52/63 (83%)</strong></td><td>10/13 (77%)</td><td>3/7 (43%)</td><td><strong>13/20 (65%)</strong></td></tr><tr><td>Miscellaneous</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Dysmorphic features</td><td>HP:0001999</td><td>30/39 (77%)</td><td>8/8 (100%)</td><td><strong>38/47 (81%)</strong></td><td>9/15 (60%)</td><td>6/9 (67%)</td><td><strong>15/24 (63%)</strong></td></tr><tr><td>Abnormal spine curvatures</td><td>HP:0010674</td><td>15/42 (36%)</td><td>+ (3/3)</td><td>18/45 (40%)</td><td>6/16 (38%)</td><td>n/a</td><td>6/16 (38%)</td></tr><tr><td>Lethality</td><td>HP:0003819</td><td>12/32 (38%)</td><td>2/29 (6%)</td><td>14/61 (23%)</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

Bold: most frequent features.

CH, compound heterozygous; DN, de novo; F, females; GDD, global developmental delay; H, homozygous; HPO, human phenotype ontology; ID, intellectual disability; IQR, interquartile range; M, males; n/a, not available.
<figure><img src="image_4.png" /></figure>

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<figure><img src="image_7.png" /><figcaption>**Figure 1** Overview of dominant and recessive ACTL6B-related disorder. A. Bar plot showing the frequency (%) of the main clinical features associated with ACTL6B-related disorder (on the left: recessive disorder; on the right: dominant disorder). B. Radar plot displaying the overlapping or distinguishable features of recessive and dominant disorder. C. Degree of global developmental delay (GDD)/intellectual disability (ID) in the disorders. D. Age span of achievement of developmental milestones in the recessive and dominant cohort compared with normal range. On the right, triangular radar plot showing the percentage of patients who achieved milestones at last evaluation.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 2** Dysmorphology, neuroimaging and epileptic findings in *ACTL6B*-related disorder. A. Facial photographs of 18 newly reported patients with *ACTL6B* recessive disorder. B. Facial photographs of 4 newly reported patients with *ACTL6B* dominant disorder. C. Boxplot displaying head circumference measurements in SD to the norm. D. Bar plot showing the frequency (%) of the main dysmorphic features. E. Bar plot showing the frequency (%) of the main neuroimaging findings. F. Epileptic phenotype of patients with recessive disorder. On the left, bar plot documenting age at onset; in the middle, sunburst chart displaying seizure types, on the right, box plots documenting seizure duration (seconds) and frequency (seizures/day).</figcaption></figure>
facial dysmorphic features observed in autosomal recessive ACTL6B-related NDD included narrow forehead/bifrontal/bitemporal narrowing (79%), full or broad nasal tip (76%), thick/full lower lip vermilion (65.5%), and broad, tall, or pointed chin (79%). Detailed facial dysmorphic features for all patients are shown in Supplemental Table 5 and tabulated using a unique Human Phenotype Ontology identification number to indicate the frequency of each feature. Supplemental Table 5 also includes patients for whom photographs were not available for assessment.

## Autosomal dominant ACTL6B-related neurodevelopmental disorder

The cohort of cases with autosomal dominant (AD) inheritance comprises 16 females and 10 males, with their age at last evaluation ranging from 1 to 29 years (median 6 years, IQR 9). Consanguinity was reported in only 2 families. Limited information was available on the prenatal/perinatal period. When gestational age at birth was provided, delivery occurred after a full-term pregnancy in 93% of the cases, with 1 baby having a preterm delivery. For those cases where birth parameters of length, weight, and occipitofrontal head circumference were provided, 3 newborns presented with normal ranges, whereas the other 3 were small for gestational age or had low birthweight, and 3 presented with microcephaly at birth (3/10, 30%). Most babies (87%) exhibited infantile hypotonia (13/15). Feeding difficulties and FTT were present in 53% and 38% of the cases, respectively. Epilepsy occurred only in 2 children (2/25, 8%). One child presented with infantile spasms at 3 months, which subsided at 18 months, unresponsive to vigabatrin, and subsequently developed focal tonic-clonic seizures with secondary generalization at 18 years. In the other affected individual, generalized seizures began at 1.6 years of age, with each episode lasting 30 seconds and occurring daily (up to 5/day). In both cases, seizures were responsive to ASMs. EEG recordings were not available in either of the cases. EEG abnormalities were reported in only 1 patient with no clinical seizures (1/8; 12.5%). Particularly, irregular, slow-wave/sharp-wave complexes and diffusely disorganized and slow background activity were documented. All affected children manifested GDD and ID (26/26). Motor delay was documented in 96% of the patients. Half of the cohort had not achieved independent sitting or walking at the last evaluation, with median ages of achievement of unsupported sitting and walking being 2 and 4 years, respectively (IQR 0.8 and 3.5). Absence of speech was reported in 81% of the children (21/26), and speech development was delayed in the remaining 5 children (19%; median age at first words is 1.5 y). When formal cognitive assessment was performed, the degree of ID ranged from moderate (n = 4), severe (n = 17) to profound (n = 3). Autistic features were present in 14 patients (67%). At the last evaluation, less than half of the patients (9/22, 41%) had microcephaly. Neurological examination showed axial hypotonia in 67% of the patients (14/21), and variable presence of hypertonia (5/20, 25%) and dystonia (4/15, 27%).

Fourteen patients with AD disorder underwent neuroimaging studies, with 12 having only radiology reports available and 2 whose imaging were independently reviewed. Brain abnormalities were detected in 86% of the cases who had neuroimaging performed and common findings included: prominence of the subarachnoid spaces (29%), hypoplastic corpus callosum (21%), brain atrophy (21%), ventricular enlargement (21%), hypomyelination (21%), and mega cisterna magna (14%) (Supplemental Table 4). Facial photographs and/or videos from 10 children across 10 families were subjected to review (Figure 2B). Based on this assessment, the most prevalent facial dysmorphic features include narrow forehead/bifrontal/bitemporal narrowing (60%), full or broad nasal tip (60%), thick/full lower lip vermilion (50%), and broad, tall, or pointed chin (70%). Detailed facial dysmorphic features for all patients are presented in Supplemental Table 5, tabulated using unique human phenotype ontology IDs to demonstrate the frequency of each feature. Supplemental Table 5 also includes patients without available photographs for assessment.

## Identification of biallelic and monoallelic ACTL6B variants delineate an allelic series

A total of 58 ACTL6B variants (n = 54 biallelic; n = 4 monoallelic) have been identified. Of those, 30 were novel and not previously reported in the literature. An overview of the molecular findings is available in Figure 3 and Supplemental Tables 6 and 7. Notably, all identified variants were consistently predicted as detrimental across a suite of in silico tools, affecting highly evolutionarily conserved residues. According to the American College of Medical Genetics classification, 35 were classified as pathogenic, 20 as likely pathogenic, and 3 as variants of uncertain significance. The biallelic variants identified in this study were found to be inherited from unaffected heterozygous parents. Specifically, among the patients, 60 inherited the variant in a homozygous state, whereas 14 patients inherited it in a compound heterozygous state. The molecular spectrum hereby described includes 24 nonsense variants (comprising 8 frameshift and 16 stop gain), 18 missense variants, 7 splicing variants, and 5 deletions.

Remarkably, all biallelic variants were either absent or found at very low allele frequencies in multiple variant frequency databases, ranging from 0.0 to 0.00002, (refer to Supplemental Table 6 for details). Notably, 6 variants were observed in more than 1 family. Particularly, variants p.(Phe147del) and p.(Ter427AspextTer33) were found in 8 and 4 independent families from different countries, respectively. This underscores the global relevance of these specific variants in the context of ACTL6B-disorders. These variants were divided into distinct groups based on their consequence, including missense, in-frame deletion, truncating, frameshift, and pre-mRNA splicing. Comprehensive comparisons were conducted across these genotypic groups and within individual variants in terms of clinical features. No evident genotype-to-phenotype correlation was identified in the recessive cohort (Figure 3). Four monoallelic
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<figure><img src="image_11.png" /><figcaption>F</figcaption></figure>

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<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>**Figure 3** Genetic findings and genotype-phenotype correlations. A. Schematic drawing illustrating the gene structure of ACTL6B and the location of the biallelic and monoallelic variants identified. Green pentagon: missense variant. Red square: stop gain. Purple triangle: splice variant. Blue diamond: in-frame deletion. Gray octagon: frameshift. B. Ribbon representation of a 3-dimensional model of ACTL6B obtained with AlphaFold. The 4 missense variants identified in ACTL6B were modeled and are displayed in red. By analogy with actin, ATP is shown bound in the catalytic cleft of ACTL6B, and the 4 subdomains that characterize the reference actin fold are labeled (mauve-background circles). C. Partially transparent surface and ribbon representations of ACTL6B, showing the 4 missense variants in red.</figcaption></figure>
variants were detected in the cohort, and all were de novo missense variants that were absent in several genetic variant frequency databases (listed in Supplemental Table 7). Notably, 3 of the variants occurred at the residue p.Gly343 in 24 independent families, indicating this location as a recurring hotspot for mutational events within a CpG dinucleotide.²⁴ Interestingly, 2 of the variants lead to the same amino acid change p.(Gly343Arg).

## ACTL6B is a nucleolar protein required for efficient ribosome biogenesis

The subcellular distribution of endogenous ACTL6B was established by performing immunostaining in 2 distinct cell lines, namely, SH-Sy5y (neuroblastoma) and U2OS (osteosarcoma). In neuronal cells (SH-Sy5y), ACTL6B exhibited a distinctive localization pattern around the protein pescadillo ribosomal biogenesis factor 1 (PES1), forming a “ring”. PES1 is known to label the granular subcompartment (GC) of the nucleolus, which is one of its most cortical parts. The ACTL6B ring colocalized perfectly with nucleophosmin (NPM1), another component of the GC (Figure 4A). Therefore, ACTL6B is positioned at the periphery of the GC of the nucleolus, as further supported by similar observations in U2OS cells in which ACTL6B also displayed a ring-like distribution (Figure 4B). To determine whether ACTL6B plays a role in ribosome biogenesis, particularly in pre-rRNA processing, SH-Sy5y cells were transfected with 2 independent silencers (#1 and #2) targeting the mRNAs encoding ACTL6B. After a 3-day incubation, total RNA was extracted and analyzed by northern blotting using a probe detecting major pre-rRNA processing intermediates (Figure 4C). Ethidium bromide staining of the agarose gel was also conducted to visualize the large mature rRNAs (18S and 28S) (Figure 4D). Efficient protein depletion was confirmed through Western blot analysis with a specific antibody (Figure 4E).

Although the levels of large mature rRNAs were not significantly affected by ACTL6B depletion, notable inhibitions in pre-rRNA processing were observed. Specifically, there was an accumulation of the 30S pre-rRNA (indicating inhibition of cleavage at site A0 in pathway 2, see Figure 4F) and an accumulation of the 21S pre-rRNA (indicating inhibition at site C). These observed phenotypes were consistent with both silencers. Additionally,

other phenotypes were observed only with 1 of the 2 silencers (#2), including a reduction in the primary transcript, the 47S (possibly reflecting an increased turnover), and a mild reduction of 12S (suggesting inhibition at site 3' and/or increased turnover). In conclusion, ACTL6B is a protein that localizes primarily at the periphery of the nucleolus where it is important for efficient pre-rRNA processing and thus for optimal ribosomal subunit biogenesis.

## Discussion

This study provides a comprehensive overview of ACTL6B-related disorders, including those associated with rare NDD inherited as autosomal recessive (AR, biallelic) and dominant (AD, monoallelic) traits. The core phenotype observed in both AR and AD disorders manifests as moderate-to-profound GDD/ID, minimal or absent speech, and autistic features. Overall, our findings are consistent with previously reported cases and emphasize the cardinal features and clinical synopsis of the disorder. A critical distinguishing factor between AR and AD disorders lies in the presence or absence of an epileptic phenotype. Strikingly, seizures occurred in 93% of the recessive cases, 88% of which were classified as intractable seizures. In contrast, only 2 patients with monoallelic variants presented an epileptic disorder, effectively managed by ASMs. Such a marked contrast suggests a potential link to distinct pathomechanisms underlying these disorders. Unravelling the influence of these mechanisms on epilepsy's onset, progression, and response to treatment holds promise for tailoring more effective therapeutic interventions for affected individuals.

However, other clinical features exhibited a higher prevalence in the AR disorder when compared with the AD cases. Patients with AR disorder frequently presented FTT (95%) and short stature (56%), whereas FTT was less commonly observed in the AD cohort (38%), and instances of short stature were never reported.

Motor delay was documented in nearly all cases of both AR (95%) and AD (96%) disorders. Nevertheless, the severity of the delay differed between the 2 cohorts. The percentage of achievement of motor milestones (such as walking and sitting) was notably lower in the AR group (18%-27%) compared with the AD group (39%-70%). ID

ACTL6B is a subunit of the BAF chromatin remodeler related to the yeast SWI/SNF remodeler, whose 3-dimensional structure has been determined using cryo-electron microscopy. ACTL6B is most closely related to actin-related protein7 (ARP7) of yeast SWI/SNF. In the structures of yeast SWI/SNF, ARP7 makes contacts with other remodeler subunits. By analogy with ARP7, we show the surface of ACTL6B predicted to be involved in inter-subunit contacts within the BAF remodeler (yellow). D. Schematic table representation of the missense variants identified in ACTL6B and their localization/interactions. E. Sequence of ACTL6B highlighting residues undergoing missense variants (red) and residues likely to be involved in intersubunit contacts within the BAF remodeler (yellow). The diagrams in parts (D) and (E) were generated with the program PyMOL (Schrödinger, LLC). F. Phenotype-to-genotype correlations in the recessive cohort. Variants are grouped in the following categories: (1) missense, (2) in-frame deletion, (3) truncating, (4) frameshift, (5) pre-mRNA splice site, and (6) indel. Green: feature present; yellow: feature not present; blank: information not available. Blue rectangle: homozygous; blue triangle: compound heterozygous.
<figure><img src="image_16.png" /></figure>

**Figure 4** ACTL6B is a nucleolar protein required for efficient pre-rRNA processing. A. Subcellular distribution analysis of ACTL6B in SH-SY5Y cells. Cells were labeled with an antibody specific to ACTL6B (in green) and costained with an antibody specific to the nucleolar protein nucleophosmin (NPM1) or pescadillo (PES1) (in red). In the merge panels, the blue signal corresponds to the nucleoplasm (labeled with 4',6-diamidino-2-phenylindole). Scale bar, 1 µm. B. ACTL6B was immunostained (in green) with a specific antibody in U2OS cells. Blue, 4',6-diamidino-2-phenylindole. Scale bar, 1 µm. C. Pre-rRNA processing analysis upon ACTL6B depletion in SH-SY5Y cells. Cells were depleted with 25 nM siRNA for 3 days. Total RNA was extracted and separated on a denaturing gel, transferred to a nylon membrane, and processed for northern blotting with probes recognizing 5' ETS, ITS1, or ITS2 sequences. Two silencers (#1 and #2) targeting a distinct region of ACTL6B transcripts were used independently. D. Mature rRNA analysis of samples described in panel (C). Ethidium
was universally observed in all patients (100%). Absent or
limited speech was documented in the majority of cases with
AR and AD disorders. There was a notable difference in the
degree of ID, with a higher proportion of patients with
biallelic variants exhibiting profound ID (41%) compared
with the AD cohort (13%). However, both groups presented
with a spectrum of severity ranging from moderate to
profound.

During the neurological assessment, a substantial number
of patients with biallelic variants exhibited increased tendon
reflexes (69%) and hypertonia/spasticity (77%), whereas
axial hypotonia was present in nearly half of the cases
(48%). Conversely, individuals with de novo variations
predominantly showed axial hypotonia (67%), with fewer
cases presenting hypertonia or spasticity (25%) and hyper-
reflexia (44%). Dystonia was more frequent in AR disease
(54% vs 27%), whereas dyskinetic movements were
relatively more common in AD disorder (53% vs 30%).
Autistic features were consistently present in both cohorts
(62%-67%).

Neuroradiological abnormalities were observed in both
groups, although the patients with AR disorder presented
with a higher prevalence of abnormalities and additional
findings consistent with hypoplastic corpus callosum,
parenchymal volume loss/atrophy, and prominence of the
subarachnoid spaces. It is crucial to note a limitation in this
comparison due to the restricted availability of imaging for
AD cases. Only 2 MRI snapshots were accessible for in-
dependent review, both of which unveiled the same abnor-
malities found in the AR cohort.

Upon examination of the available photographs, the most
common facial features observed in both groups consist of a
narrow forehead/bifrontal/bitemporal narrowing, full or
broad nasal tip, thick/full lower lip vermilion, and broad,
tall, or pointed chin. Our evaluation indicates the absence of
a recognizable facial gestalt for either AR or AD ACTL6B-
related NDD.

This study emphasizes the cardinal and distinct features
of the 2 ACTL6B-related disorders (DEE76 and IDDDSAD),
providing a differential diagnosis for BAFopathies, which
includes various NDDs resulting from mutations in different
subunits of the BAF complex (Supplemental Figure 1,
Supplemental Table 8). Monoallelic variants in genes
encoding BAF complex subunits are linked to different
forms of syndromic intellectual development disorders,
including Coffin-Siris syndrome and Nicolais-Baraitser
syndrome. Although these disorders share commonalities
with ACTL6B-related disorders, including GDD/ID, micro-
cephaly, short stature, and autistic features, they are distin-
guishable by characteristic facial and hand/digital features.

Unlike ACTL6B-related disorders, epilepsy is usually not a
prominent feature in Coffin-Siris syndrome and Nicolais-
Baraitser syndrome, and movement disorders or dystonia
are not reported. Epilepsy is also extremely rare in the
ACTL6B dominant cohort, suggesting its rarity in dominant
BAFopathies.

Another autosomal recessive BAFopathy, involving
biallelic variants in SMARCD2, has been reported. How-
ever, its distinct manifestations, centered around recurrent
infections and hematologic abnormalities, set it apart from
ACTL6B-related disorders. Although GDD/ID is observed,
limited knowledge about its association with neuro-
development complicates establishing a precise differential
diagnosis. The nBAF complex plays a crucial role in various
neurodevelopmental processes, influencing neurogenesis in
terms of both cell proliferation and differentiation, cell
migration, maturation, and integration of neurons. Recent
studies have shed light on the involvement of the neuronal
BAF complex in development of ASD development. Path-
ogenic variants in genes encoding BAF complex subunits,
such as ARID1B, SMARCA2, SMARCC1(HGNC:11104),
and SMARCA4, have been linked to an elevated risk of
ASD. Autistic features were frequently observed in both AR
and AD cases of ACTL6B-related disorders, providing
additional evidence of the connection between nBAF com-
plex dysfunction and ASD. However, the intricate rela-
tionship between the 2 remains not fully understood,
warranting further research to elucidate the underlying
mechanisms.

Ribosome biogenesis dysfunction has been linked to
tissue-specific disorders affecting primarily the brain, the
blood, and the bones.²⁶ Most of these diseases are deeply
rooted in the embryonic development, and the brain disor-
ders are no exception. It was notably shown using different
animal models (frog, mouse, etc.) that optimal ribosome
biogenesis is instrumental for proper maturation of neural
crest cells, which are important for the development of the
bone structure of the head and of the peripheral nervous
system, among others.²⁷,²⁸ The importance of ribosome
biogenesis in neurological disorders was also illustrated by
mutations in a gene encoding the methyltransferase
METTL5-TRM112 acting on the small ribosomal subunit
rRNA, the 18S. The absence of this methyltransferase leads
to production of ribosomes lacking a single methyl group.²⁹
This could be considered a rather insignificant difference
considering the ribosome is a multi-mega-kDa nanoma-
chine, yet it is enough to cause GDD, microcephaly and
associated manifestations,³⁰ including ID and unbalanced
walking, which could be recapitulated in a mutant mouse
and fly, respectively.³¹,³²

bromide (EtBr) staining revealed the steady-state levels of mature 18S and 28S rRNAs. E. Western blot analysis confirming efficient protein depletion. The antibody used are described in the Materials and Methods. As control for loading, the blot was probed for β-actin. F. Pre-rRNA processing pathway depicting major intermediates and probes used. See https://doi.org/10.1016/j.biochi.2012.02.001<sup>25</sup> for details.
Our study expands the genotypic spectrum of both *ACTL6B*-associated disorders, introducing 26 biallelic and 1 monoallelic variant that were not previously reported. All biallelic variants are anticipated to result in LoF, according to Bell et al,⁵ and are distributed across the protein in intradomain structured regions.³³ Conversely, monoallelic variants affecting the amino acid residue p.Gly343 are expected to exert DN effects, influencing the affinity to nucleosomal DNA by introducing a side chain upon mutation variation to arginine or tryptophan.⁵,³³ *ACTL6B* is among the first genes of the multisubunit protein BAF complex for which both recessive and dominant variants are pathogenic, with distinct consequences on protein function (LoF vs DN). Pathogenic monoallelic variants in other BAF complex genes are predicted to result in either LoF (*ARID1A*, *ARID1B*, *ARID2*, *SMARCC2*, and *SMARCD1*) or DN/GoF (*SMARCA4*, *SMARCE1*, and *SMARCB1*). Furthermore, disruptions in various structural hubs of the BAF complex (eg, histone binding, catalytic ATPase, and actin-related protein- module) might contribute to the variability in symptoms and severity observed in different BAFopathies.³³

This study also investigates the role of ACTL6B in ribosome biogenesis and provides a likely molecular explanation for *ACTL6B*-related disorders. ACTL6B had been assigned tentatively to the nucleolus,¹¹ a multilayer biomolecular condensate where ribosome biogenesis is initiated. Our study has confirmed the localization of ACTL6B in the nucleolus and has revealed that the protein is enriched in a ring at the periphery of the organelle. In vitro, experiments on model cells demonstrated that mature rRNAs levels were not grossly affected upon ACTL6B depletion. However, the protein is required for efficient pre-rRNA processing at specific cleavage steps; therefore, its dysfunction does compromise important steps of ribosome biogenesis. Ribosome biogenesis disorders, collectively referred to as ribosomopathies, designate an emerging class of syndromes, including neurological disorders, caused by pathogenic variants in a ribosomal protein or a ribosomal assembly factor. The association between ribosome biogenesis dysfunction and NDDs has only started to unveil. Future research is warranted to establish fully connections between these disorders and ribosomes. With the evidences presented here, we suggest to include *ACTL6B*-associated disorders in the expanding spectrum of ribosomopathies (Supplemental Table 9).

Although our study provides novel insights into *ACTL6B*-related disorders, it is important to acknowledge the potential limitations arising from our relatively small and homogenous sample size. This constraint may not fully encompass the genetic and phenotypic diversity present within the broader population affected by these conditions. Nevertheless, our work offers a comprehensive overview of *ACTL6B*-related disorders, including the disease spectrum, genotype-to-phenotype relationships, and differential diagnosis based on mutational status. These insights contribute valuable information for improving clinical diagnosis and guiding therapeutic approaches. Additionally, our study proposes a probable molecular explanation for the pathology: inefficient ribosome

biogenesis. This process has been previously linked to defects in the brain (neurodevelopmental and neurological issues) and bones (skeletal development, short stature, etc). Therefore, our findings improve the understanding of the *ACTL6B*-related syndrome and contribute to the expanding knowledge of diseases associated with ribosome biogenesis dysfunction.

## Data Availability

The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. For families R9, R16 and D5, data are available in the National Genomics Research Library v5.1, Genomics England (https://doi.org/10.6084/m9.fishshare.4530893/7.2020).

## Acknowledgments

We are grateful for the important support from patients and families, our UK and international collaborators, brainbanks and biobanks, and grateful for essential funding from The Wellcome Trust, The MRC, The MSA Trust, The National Institute for Health Research, University College London Hospitals Biomedical Research Centre (NIHR-BRC), The Michael J Fox Foundation (MJFF), The Fidelity Trust, Rosetrees Trust, Dolby Family fund, Alzheimer's Research UK (ARUK), MSA Coalition, Parkinson's disease society, Parkinson's Foundation, The Guarantors of Brain, Cerebral Palsy Alliance, FARA, EAN, Victoria Brain Bank, the NIH NeuroBioBank, Queen Square BrainBank, The MRC Brainbank Network.

## Funding

This study was supported by the Wellcome Trust (WT093205MA and WT104033AIA to H.H.), Medical Research Council (H.H.), European Community's Seventh Framework Programme (FP7/2007-2013, under grant agreement No. 2012-305121 to H.H.), the National Institute for Health Research, University College London Hospitals, Biomedical Research Centre, and Fidelity Foundation. This study was also supported in part by US National Institutes of Health R35NS105078 to J.R.L., and UM1 HG011758 to J.E.P. and J.R.L., and Muscular Dystrophy Association (MDA) (512848 to J.R.L.). D.M. was supported by a Medical Genetics Research Fellowship Program through the United States National Institute of Health (T32 GM007526-42). D.P. is supported by International Rett Syndrome Foundation, Grant/Award Number: #3701-1. J.E.P. was supported by NHGRI K08 HG008986. T.M. was supported by the Uehara Memorial Foundation. R.D. was supported by NIH grant R01 GM073791. Research in the Lab of D.L.J.L. was supported by the Belgian Fonds de la Recherche
Scientifique (F.R.S./FNRS), EOS [CD-INFLADIS], Région Wallonne (SPW EER) Win4SpinOff [RIBOGENESIS], the COST action TRANSLACORE (CA21154), and the European Joint Programme on Rare Diseases (EJP-RD) RiboEurope and DBAGeneCure. M.M. and P.T. were supported by NW24-06-00083.

## Author Contributions

Conceptualization: E.C., T.Q., C.R., S.E., D.L., H.H., R.M.; Data Curation: E.C., D.M., M.S., A.R., P.S., A.M.D.L., R.M.; Formal Analysis: E.C., T.Q., D.L., R.M.; Funding Acquisition: H.H., J.R.L., J.E.P., D.M., D.P., R.D., P.S., T.M.; Methodology: E.C., T.Q., C.R., M.S., R.D., A.M.D.L., S.E., D.L., R.M.; Supervision: D.L., H.H., R.M.; Writing-original draft: E.C., T.Q., M.S., R.D., A.M., D.L., R.M.; Writing-review and editing: all authors.

## Ethics Declaration

Individuals (and/or their legal guardians) recruited in a research setting gave informed consent for their research participation. Those individual research studies received approval from the Review Boards and Bioethics Committees at University College London Hospital (project 06/ N076) and the other institutions involved in this study. Permission for inclusion of their anonymized medical data in this cohort, including photographs, was obtained using standard forms at each local site by the responsible referring physicians.

## Conflict of Interest

Sureni V. Mullegama and Amber Begtrup are employees of GeneDx. Lee Hane is an employee at 3billion. Christian Beetz is an employee at Centogene. All other authors declare no conflicts of interest.

## Additional Information

The online version of this article (https://doi.org/10.1016/j.gim.2024.101251) contains supplemental material, which is available to authorized users.

## Affiliations

de Bruxelles (ULB), Biopark campus, Gosselies, Belgium; ³Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, Genoa, Italy; ⁴Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; ⁵Department of Pediatrics, Faculty of Medicine, Kuwait University, Safat, Kuwait; ⁶Clinical Genetics Department, Human Genetics and Genome Research Institute, National Research Centre, Cairo, Egypt; ⁷UK National Paediatric Ataxia Telangiectasia Clinic, Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom; ⁸Nottingham Clinical Genetics Service, Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom; ⁹Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; ¹⁰Palindrome, Isfahan, Iran; ¹¹Medical Molecular Genetics Department, Human Genetics and Genome Research Institute, National Research Centre Cairo, Egypt; ¹²Human Genetics Department, Medical Research Institute, Alexandria University, Egypt; ¹³Unité Fonctionnelle 6254 d'Innovation en Diagnostique Génomique des Maladies Rares, Pôle de Biologie, CHU Dijon Bourgogne, Dijon, France; ¹⁴INSERM UMR1231 GAD, Dijon, France; ¹⁵Service de génétique médicale, CHU de Nantes, Nantes, France; ¹⁶Institut du thorax, INSERM, CNRS, UNIV Nantes, Nantes, France; ¹⁷Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic; ¹⁸Department of Paediatric Neurology, Children's Hospital and Institute of Child Health, Multan, Pakistan; ¹⁹College of Medicine/University of Baghdad, Unit of Pediatric Neurology, Children Welfare Teaching Hospital, Baghdad, Iraq; ²⁰Izmir Katip Çelebi University Tepecik Training and Research Department of Pediatric Neurology, Izmir, Turkey; ²¹University of Health Sciences, Gülhane Faculty of Medicine, Department of Child Neurology, Ankara, Turkey; ²²Unité Fonctionnelle d'Innovation diagnostiques des maladies rares, FHU TRANSLAD, CHU Dijon Bourgogne, Dijon, France; ²³INSERM-Université de Bourgogne UMR1231 GAD "Génétique des Anomalies du Développement" Dijon, France; ²⁴Département de Génétique Médicale, Maladies rares et Médecine Personnalisée, et Centre de Référence Anomalies du Développement et Syndromes Malformatifs, CHRU de Montpellier, Montpellier, France; ²⁵Genetic Center, Akron Children's Hospital, Akron, OH; ²⁶Department of Medical Genetics, Samsun University, Faculty of Medicine, Samsun, Turkey; ²⁷Laboratory of Medical Genetics, Translational Cytogenomics Research Unit, Bambino Gesù Children Hospital, IRCCS, Rome, Italy; ²⁸Department of Neurosciences, Unit of Neurorehabilitation, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy; ²⁹Aix-Marseille Univ, APHM, department of Pediatrics Neurology. Timone children Hospital. Marseille, France; ³⁰Aix Marseille Univ, Inserm, MMG, Marseille, France Service de Génétique Médicale, AP-HM, Hôpital de La Timone, Marseille, France; ³¹Department of Clinical Genetics, Erasmus MC,

<footnote n="1">¹Department of Neuromuscular Diseases, University College London, Queen Square, Institute of Neurology, London, United Kingdom; ²RNA Molecular Biology, Fonds de la Recherche Scientifique (F.R.S./FNRS), Université libre</footnote>
Rotterdam, The Netherlands; ³²Institute of Human Genetics, University of Leipzig Medical Center, Leipzig, Germany; ³³Sulaiman Al Habib Hospital - Olaya Medical Complex-Riyadh, Saudi Arabia; ³⁴Department of Paediatric and Child Health, Aga Khan University Hospital, Karachi, Pakistan; ³⁵Maternal, Fetal and Neonatal Research Center, Family Health Research Institute, Tehran University of Medical Sciences, Tehran, Iran; ³⁶Genetics Ward, Yas Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran; ³⁷Department of Medical Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; ³⁸Division of Genetics, Arnold Palmer Hospital for Children, Orlando Health, Orlando, FL; ³⁹Genomic Medicine Center, Children's Mercy Hospital, Kansas City, MO; ⁴⁰Kansas City School of Medicine, University of Missouri, Kansas City, MO; ⁴¹Department of Pathology and Laboratory Medicine, Children's Mercy Hospital, Kansas City, MO; ⁴²The Community Health Clinic, Shipshewana, IN; ⁴³Department of Genetics, Southern California Permanente Medical Group, Fontana, CA; ⁴⁴Clinical Genetic Services, Department of Pediatrics, NYU Grossman School of Medicine, New York, NY; ⁴⁵Clinical Genetics, NYU Orthopedic Hospital, New York, NY; ⁴⁶Clinical Genetics, Center for Children, Hassenfeld Children's Hospital, New York University, New York, NY; ⁴⁷Laboratory of Medical Genetics, Medical School, National and Kapodistrian University of Athens, St. Sophia's Children's Hospital, Athens, Greece; ⁴⁸Pardis Pathobiology and Genetics Laboratory, Mashhad, Iran; ⁴⁹Department of Medical Genetics and Molecular Medicine, School of Medicine, Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran; ⁵⁰Service de Génétique Clinique, CHU Lille, Lille, France; ⁵¹Department of Human Genetics, Radboudumc University Medical Center, Nijmegen, The Netherlands; ⁵²Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, The Netherlands; ⁵³Department of Cell and Molecular Biology and Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran; ⁵⁴Genetics of Non-Communicable Disease Research Center, Zahedan University of Medical Sciences, Zahedan, Iran; ⁵⁵Centre Mohamed VI for Research and Innovation (CM6RI) and University Mohamed VI for Health Science (UM6SS), Benguerir, Morocco; ⁵⁶Molecular Genetics Laboratory, Istishari Arab Hospital, Ramallah, Palestine; ⁵⁷Faculty of Medical Sciences, Mohammed 6 Polytechnic University of Benguerir, Ben Guerir, Morocco; ⁵⁸Veltischev Research and Clinical Institute for Pediatrics of the Pirogov, Russian National Research Medical University, Ministry of Health of Russian Federation, Moscow, Russia; ⁵⁹Mental Health Research Center, Moscow, Russia; ⁶⁰Research Centre for Medical Genetics, Moscow, Russia; ⁶¹Paediatrics Wah Medical College NUMS, Wah Cantonment, Punjab, Pakistan; ⁶²University of Chicago Medicine, University of Chicago, Chicago, IL; ⁶³Department of Paediatrics and Genetics, Universidade Federal de Paraíba, Joao Pessoa,

Paraíba, Brazil; ⁶⁴Cellular, Molecular and Genetics Research Center, Isfahan University of Medical Sciences, Isfahan, Iran; ⁶⁵Medical Genetics Research Center of Genome, Isfahan University of Medical Sciences, Isfahan, Iran; ⁶⁶Department of Pediatric Neurology, Neo Clinic Children's Hospital, Jaipur, India; ⁶⁷Department of Human Genetics, The University of Chicago, Illinois; ⁶⁸Department of Pediatric Neurology, Indira Gandhi Institute of Child Health, Bengaluru, India; ⁶⁹Department of Pediatric Neurology - Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), Boa Vista, Recife, Brazil; ⁷⁰Oxford Genetics Laboratories, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom; ⁷¹Clinical Genetics service, Northampton General Hospital, Northampton, United Kingdom; ⁷²Department of Pediatrics, Fayoum University Hospitals, Fayoum, Egypt; ⁷³Instituto de Medicina Integral Prof. Fernando Figueira, Centro de Terapias Cetogênicas do IMIP, Recife PE, Brazil; ⁷⁴Department of Pediatric Gastroenterology Hepatology and Genetic diseases Children's Hospital and University of Child Health Sciences Lahore, Pakistan; ⁷⁵Al-Quds University, Jerusalem, Palestine; ⁷⁶PRCS hospital, Hebron, Palestine; ⁷⁷Department of Genetics, Hadassah Medical Center, Jerusalem, Israel Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel; ⁷⁸CENTOGENE GmbH, Rostock, German; ⁷⁹Mendelics Genomic Analysis, São Paulo, Brazil; ⁸⁰Hospices Civils de Lyon, Service de Génétique, Bron, France; ⁸¹Pathophysiology and Genetics of Neuron and Muscle (PGNM, UCBL - CNRS UMR5261 - INSERM U1315), Université Claude Bernard Lyon 1, Lyon, France; ⁸²Department of Neuropediatric, University Hospital of Lyon, Lyon, France; ⁸³Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London, United Kingdom; ⁸⁴Department of Pediatrics, Vanderbilt Kennedy Center, Vanderbilt University Medical Center, Nashville, TN; ⁸⁵GeneDx, Gaithersburg, MD; ⁸⁶Section of Pediatric Neurology and Developmental Neuroscience, Department of Pediatrics, Baylor College of Medicine, Houston, TX; ⁸⁷Clinical Research Centre, Sunway Medical Centre, Malaysia; ⁸⁸Department of Paediatric Neurology, Children Hospital and Institute of Child Health, Faisalabad, Pakistan; ⁸⁹Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom; ⁹⁰West Midlands Clinical Genetics Service, Birmingham Women's Hospital, Birmingham, United Kingdom; ⁹¹Division of Medical Genetics, 3billion, Inc, Seoul, South Korea; ⁹²Department of Pediatrics, Faculty of Medicine, Sohag University, Sohag, Egypt; ⁹³Laboratório Mendelics, Department of Genetic, São Paulo, Brazil; ⁹⁴Neurology Unit, Department of Pediatrics, Faculty of Medicine, Alexandria University, Egypt; ⁹⁵Genomic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; ⁹⁶Department of Paediatrics, Murdoch Children's Research Institute and University of Melbourne, Royal Children's Hospital, Melbourne, Australia; ⁹⁷Department of Pediatrics, Boston Children's
Hospital, Harvard Medical School, Boston, MA; ⁹⁸Department of Medical Genetics, Next Generation Genetic Polyclinic, Mashhad, Iran; ⁹⁹Molecular and Clinical Sciences Institute, St. George's, University of London, Cranmer Terrace, London, United Kingdom; ¹⁰⁰Innovative Medical Research Center, Mashhad Branch, Islamic Azad University, Mashhad, Iran; ¹⁰¹CHU Sainte-Justine Research Center, Montreal, QC, Canada; ¹⁰²Department of Pediatrics, Faculty of Medicine, Université de Montréal, Montreal, QC, Canada; ¹⁰³Department of Translational Genomics, Center for Genomic Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia; ¹⁰⁴NIHR Biomedical Research Centre, Centre for Human Genetics, University of Oxford, Oxford, United Kingdom; ¹⁰⁵Department of Neurosciences, University of California, San Diego, La Jolla, CA; ¹⁰⁶Rady Children's Institute for Genomic Medicine, San Diego, CA; ¹⁰⁷Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX; ¹⁰⁸Department of Pediatrics, Baylor College of Medicine, Houston, TX; ¹⁰⁹Pediatric Neurology and Muscular Diseases Unit, IRCCS Istituto Giannina Gaslini, Genova, Italy; ¹¹⁰Department of Radiology, Neuroradiology Section, Kingston Health Sciences Centre, Queen's University Faculty of Health Sciences, Kingston, Ontario, Canada

## References

1. Olave I, Wang W, Xue Y, Kuo A, Crabtree GR. Identification of a polymorphic, neuron-specific chromatin remodeling complex. *Genes Dev.* 2002;16(19):2509-2517. http://doi.org/10.1101/gad.992102

2. Wu JI, Lessard J, Olave IA, et al. Regulation of dendritic development by neuron-specific chromatin remodeling complexes. *Neuron*. 2007;56(1):94-108. http://doi.org/10.1016/j.neuron.2007.08.021

3. Lessard J, Wu JI, Ranish JA, et al. An essential switch in subunit composition of a chromatin remodeling complex during neural development. *Neuron*. 2007;55(2):201-215. http://doi.org/10.1016/j.neuron.2007.06.019

4. Sokpor G, Xie Y, Rosenbusch J, Tuoc T. Chromatin remodeling BAF (SWI/SNF) complexes in neural development and disorders. *Front Mol Neurosci.* 2017;10:243. http://doi.org/10.3389/fnmol.2017.00243

5. Bell S, Rousseau J, Peng H, et al. Mutations in ACTL6B cause neurodevelopmental deficits and epilepsy and lead to loss of dendrites in human neurons. *Am J Hum Genet.* 2019;104(5):815-834. http://doi.org/10.1016/j.ajhg.2019.03.022

6. Sekiguchi F, Tsurusaki Y, Okamoto N, et al. Genetic abnormalities in a large cohort of Coffin-Siris syndrome patients. *J Hum Genet.* 2019;64(12):1173-1186. http://doi.org/10.1038/s10038-019-0667-4

7. Santen GWE, Aten E, Vulto-van Silfhout AT, et al. Coffin-Siris syndrome and the BAF complex: genotype-phenotype study in 63 patients. *Hum Mutat.* 2013;34(11):1519-1528. http://doi.org/10.1002/humu.22394

8. Chen CA, Lattier J, Zhu W, et al. Retrospective analysis of a clinical exome sequencing cohort reveals the mutational spectrum and identifies candidate disease-associated loci for BAFopathies. *Genet Med.* 2022;24(2):364-373. http://doi.org/10.1016/j.gim.2021.09.017

9. Cappuccio G, Sayou C, Tanno PL, et al. De novo SMARCA2 variants clustered outside the helicase domain cause a new recognizable syndrome with intellectual disability and blepharophimosis distinct from Nicolaides-Baraitser syndrome. *Genet Med.* 2020;22(11):1838-1850. http://doi.org/10.1038/s41436-020-0898-y

10. Abdul-Rahman O. Nicolaides-Baraitser syndrome. In: Adam MP, Mirzaa GM, Pagon RA, et al., eds. *GeneReviews*. Seattle: University of

Washington; 2015. Accessed October 23, 2024. https://www.ncbi.nlm.nih.gov/books/NBK321516/

11. Peron A, Bradbury K, Viskochil DH, Dias C. In: *BCL11A-related intellectual disability*. Seattle: GeneReviews. University of Washington; 2019. Accessed October 23, 2024. https://www.ncbi.nlm.nih.gov/books/NBK547048/

12. Lessel D, Gehbauer C, Bramswig NC, et al. BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells. *Brain*. 2018;141(8):2299-2311. http://doi.org/10.1093/brain/awy173

13. Fichera M, Failla P, Saccuzzo L, et al. Mutations in ACTL6B, coding for a subunit of the neuron-specific chromatin remodeling complex nBAF, cause early onset severe developmental and epileptic encephalopathy with brain hypomyelination and cerebellar atrophy. *Hum Genet.* 2019;138(2):187-198. http://doi.org/10.1007/s00439-019-01972-3

14. Maddirevula S, Alzahrani F, Al-Owain M, et al. Autozygome and high throughput confirmation of disease genes candidacy. *Genet Med.* 2019;21(3):736-742. http://doi.org/10.1038/s41436-018-0138-x

15. Yüksel Z, Yazol M, Gümüş E. Pathogenic homozygous variations in ACTL6B cause DECAM syndrome: developmental delay, epileptic encephalopathy, cerebral atrophy, and abnormal myelination. *Am J Med Genet A.* 2019;179(8):1603-1608. http://doi.org/10.1002/ajmg.a.61210

16. Wenderski W, Wang L, Krokhotin A, et al. Loss of the neural-specific BAF subunit ACTL6B relieves repression of early response genes and causes recessive autism. *Proc Natl Acad Sci U S A.* 2020;117(18):10055-10066. http://doi.org/10.1073/pnas.1908238117

17. Han X, Deng J, Chen C, et al. Developmental and epileptic encephalopathy 76: case report and review of literature. *Children (Basel)*. 2022;9(12):1967. http://doi.org/10.3390/children9121967

18. Stenström L, Mahdessian D, Gnann C, et al. Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic protein disorder. *Mol Syst Biol.* 2020;16(8):e9469. http://doi.org/10.15252/msb.20209469

19. Hetman M, Slomnicki LP. Ribosomal biogenesis as an emerging target of neurodevelopmental pathologies. *J Neurochem.* 2019;148(3):325-347. http://doi.org/10.1111/jnc.14576

20. Sobreira N, Schiettecatte F, Valle D, Hamosh A. GeneMatcher: a matching tool for connecting investigators with an interest in the same gene. *Hum Mutat.* 2015;36(10):928-930. http://doi.org/10.1002/humu.22844

21. Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. *Nature*. 2021;596(7873):583-589. http://doi.org/10.1038/s41586-021-03819-2

22. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of coot. *Acta Crystallogr D Biol Crystallogr.* 2010;66(4):486-501. http://doi.org/10.1107/S0907444910007493

23. Abramoff M, Magalhaes P, Ram S, et al. Image processing with Image. *J Biophot Int.* 2004;11(7):36-42.

24. Hodgkinson A, Eyre-Walker A. Variation in the mutation rate across mammalian genomes. *Nat Rev Genet.* 2011;12(11):756-766. http://doi.org/10.1038/nrg3098

25. Mullineux ST, Lafontaine DLJ. Mapping the cleavage sites on mammalian pre-rRNAs: where do we stand? *Biochimie.* 2012;94(7):1521-1532. http://doi.org/10.1016/j.biochi.2012.02.001

26. Farley-Barnes KI, Ogawa LM, Baserga SJ. Ribosomopathies: old concepts, new controversies. *Trends Genet.* 2019;35(10):754-767. http://doi.org/10.1016/j.tig.2019.07.004

27. Delhermite J, Tafforeau L, Sharma S, et al. Systematic mapping of rRNA 2'-O methylation during frog development and involvement of the methyltransferase fibrillarin in eye and craniofacial development in Xenopus laevis. *PLoS Genet.* 2022;18(1):e1010012. http://doi.org/10.1371/journal.pgen.1010012

28. Dixon J, Jones NC, Sandell LL, et al. Tcof1/Treacle is required for neural crest cell formation and proliferation deficiencies that cause craniofacial abnormalities. *Proc Natl Acad Sci U S A.* 2006;103(36):13403-13408. http://doi.org/10.1073/pnas.0603730103
29. van Tran N, Ernst FGM, Hawley BR, et al. The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112. *Nucleic Acids Res.* 2019;47(15):7719-7733. http://doi.org/10.1093/nar/gkz619

30. Richard EM, Polla DL, Assir MZ, et al. Bi-allelic variants in METTL5 cause autosomal-recessive intellectual disability and microcephaly. *Am J Hum Genet.* 2019;105(4):869-878. http://doi.org/10.1016/j.ajhg.2019.09.007

31. Leismann J, Spagnuolo M, Pradhan M, et al. The 18S ribosomal RNA m6A methyltransferase Mettl5 is required for normal walking behavior

in Drosophila. *EMBO Rep.* 2020;21(7):e49443. http://doi.org/10.15252/embr.201949443

32. Wang L, Liang Y, Lin R, et al. Mettl5 mediated 18S rRNA N6-methyladenosine (m6A) modification controls stem cell fate determination and neural function. *Genes Dis.* 2022;9(1):268-274. http://doi.org/10.1016/j.gendis.2020.07.004

33. Valencia AM, Sankar A, van der Sluijs PJ, et al. Landscape of mSWI/SNF chromatin remodeling complex perturbations in neuro-developmental disorders. *Nat Genet.* 2023;55(8):1400-1412. http://doi.org/10.1038/s41588-023-01451-6