Biallelic variants in CELSR1 cause brain malformations, neurodevelopmental disorders and epilepsy in humans

Domain: biology
Published: 2026-01-13
Editorial outcome: major_revision

Abstract
The CELSR1 gene is a core component of the tissue/planar cell polarity signaling pathway. It encodes a developmentally regulated protein that belongs to the adhesion G protein-coupled receptors. Herein we describe seven subjects, from five unrelated families, featuring a neurodevelopmental disorder associated with biallelic CELSR1 variants. The main phenotypic features of this disorder are different types of brain malformations (including pachygyria, periventricular nodular heterotopia, abnormal corpus callosum, white matter abnormalities, hypoplasia of brainstem and cerebellum), variable degrees of neurodevelopmental delay and intellectual disability, behavioral disorders, and, in some subjects, epilepsy. Using whole exome sequencing, we identify five compound heterozygous variants and one homozygous variant of CELSR1 in these subjects. We infer the pathogenicity and functional effects of these variants through bioinformatic analysis, protein modelling and prediction tools. To further characterize the effects of mutant CELSR1, we generate Celsr1 knockout mice, which exhibit partial agenesis of the corpus callosum, periventricular heterotopia and irregular shape of the ventricular/subventricular zone, enlarged lateral ventricles with a fully penetrant phenotype, and increased susceptibility to seizures. These findings emphasize the importance of CELSR1 in several polarity-dependent processes during embryonic and postnatal development.

Introduction
In humans, the central nervous system (CNS) starts to develop early in embryonic life, and its development continues for years after birth. Neurodevelopmental delay (NDD) is a group of heterogeneous childhood conditions with high clinical heterogeneity and affects ~1–3% of children worldwide1. Features of NDD are impairment in cognition, language, behavior, and/or motor skills (such as impaired gross motor abilities and fine-motor coordination), resulting from abnormal brain development. Intellectual disability (ID), autism spectrum disorder (ASD), and attention deficit/hyperactivity disorder (ADHD) are common comorbidities associated with NDD2. ID is defined as a disorder with onset during the developmental period that includes both intellectual and adaptive functioning deficits in conceptual, social, and practical domains3. It affects approximately one to four percent of the population in developed countries4. NDD and/or ID are common manifestations of various genetic neurodevelopmental disorders, and they are often associated with epilepsy, ASD, and ADHD, which are the three most frequent neurodevelopmental comorbidities in patients with NDD with a known genetic cause5,6. Developmental epileptic encephalopathies (DEE) are a group of NDD/ID conditions in which uncontrolled epileptic activity plays a deleterious role in the regression or further slowing of neurodevelopment7. Indeed, in DEE, epileptic activity “per se” can contribute to severely impair cognitive and behavioral development, above what is expected from the underlying pathology, and the developmental consequences may improve with the amelioration of the epileptiform activity8. Encompassed in the group of DEEs, encephalopathy related to status epilepticus during sleep (ESES), recently labeled as DEE with spike-wave activation during sleep (DEE-SWAS) (hence the label ESES/DEE-SWAS that we use in this paper), refers to epileptic conditions, often with a genetic cause, that are characterized by various combinations of cognitive, language, behavioral, and motor disturbances whose onset or worsening is associated with the appearance of a marked enhancement of EEG epileptiform activities during sleep9.

To date, over 1300 causative genes and 1100 candidate genes related to NDD/ID pathogenesis have been identified, and the number continues to grow annually10,11. Making a correct and prompt diagnosis of a genetic neurodevelopmental disorder is important to end a lengthy, resource-consuming diagnostic odyssey, to improve treatment, to inform management even in the absence of established curative or disease-specific therapies, and to provide counseling to patients and families.

The CELSR1 gene (also known as FMI2, OMIM*604523), located on chromosome 22q13.31 in humans and chromosome 15 in mice, encodes a developmentally regulated protein that belongs to the adhesion G protein-coupled receptors (adhesion GPCR)11–14. The CELSR1 protein is a core component of the tissue/planar cell polarity (PCP) signaling, which governs multiple cell polarization-dependent processes such as convergent extension, oriented cell division, and directional migration13–16. It is characterized by a large extracellular domain that contains nine contiguous cadherin repeats, eight epidermal growth factor (EGF)-like domains intermixed by three laminin A G-type (LAG) repeats, followed by a GAIN (G protein-coupled receptor (GPCR) autoproteolysis-inducing domain) with the proteolytic site (GPS)17, (WWW.NCBI.NLM.NIH.GOV/GENE/9620). The N-terminus is followed by seven transmembrane domains, and an intracellular C-terminal tail with no clear motives. CELSR1 is believed to mediate homotypic interactions and connect the extracellular matrix to the cytoskeleton. It is involved in morphogenesis of several organs and systems including the kidney, lung, skin, prostate, salivary gland, trachea, thyroid, urogenital tract, and lymphatic system12,17–22. Variants in the CELSR1 gene have been linked to congenital defects in the vestibulocochlear system23,24, heart25, kidney26, and lung21. A link between CELSR1, lymphatic system development, and lymphedema has also been documented for heterozygous LOF variants22,27–29. In the nervous system, CELSR1 is highly expressed in neuroepithelial cells during the prenatal period, as well as in neural progenitors and ependymal cells in the postnatal brain20,30 (HUMAN.BRAIN-MAP.ORG). Pathogenic CELSR1 variants were associated with neural tube closure defects and caudal agenesis23,31–35.

In mice, CELSR1 plays a crucial role in the neurogenic switch during histogenesis of the cerebral cortex. In the absence of CELSR1, cortical progenitor cells undergo more proliferative divisions at the expense of neuron production, causing cortical hypoplasia, learning and memory deficits, and autistic-like behavior36. In the developing rhombencephalon, CELSR1 is involved in the directionality of tangential migration of facial nerve motor neurons37. CELSR1 is also key to ependymal cilia polarization and ciliary beats, and its loss severely disrupts the circulation of the cerebrospinal fluid and results in hydrocephalus30.

Here, we report in humans a distinctive neurodevelopmental disorder encompassing various types of brain malformations, NDD, ID, epilepsy, dysmorphic features, and psychiatric/behavioral comorbidities, associated with compound heterozygous or homozygous variants in CELSR1. We correlate this with Celsr1 knockout (KO) mice analysis, which recapitulates the human findings and provides insights into the cellular mechanisms that underlie morphological and behavioral defects.

Results
Patient’s data
Clinical characteristics. We collected seven individuals (five females, two males; age range: 1 month–15 years) with deleterious biallelic CELSR1 variants from five unrelated families; family pedigrees of patients #1 and #2 and of patient #4 are shown in Supplementary Fig. 1. Main clinical information of the affected individuals is summarized in Table 1 (detailed clinical information of patients are reported in Supplementary Notes). Four patients were born at term, three were born moderate to late preterm, after an uneventful pregnancy. One patient died at 1 month of age because of the severity of the clinical picture. Patients #1 and #2 are siblings of related cousins, and there is a strong recurrence of intellectual disability in the family—in five cousins—and one first-degree cousin affected by epilepsy not further specified (Supplementary Fig. 1). The parents of patient #4 are also related and two of his cousins (two sons of related parents) have a neurodevelopmental disorder, not further specified.

Behavior and neurodevelopment. A neurodevelopmental delay, ranging from mild to severe, was observed in all assessable patients. Three patients (#1, #2, and #5) were mildly impaired and attended a school for children with special needs. A mild language delay that improved over time was reported in patients #1, #2 and #5. Patients #3, #4, and #6 displayed a moderate neurodevelopmental delay, with a lack of verbal communication, expressing themselves only by single words. Attention/deficit hyperactivity disorders (ADHD) were reported in patients #2, #3, and #4. In addition, patients #3 and #4 were affected by auto- and hetero-aggressive and autistic features. Two monozygotic twin patients (#5 and #6) had a diagnosis of ASD and had an avoidant restrictive food disorder. The last patient (#7) displayed an extremely severe clinical picture with a severely depressed reactivity to external stimuli and no active movements immediately after delivery, with no spontaneous breathing and swallowing reflex, that led to death at 1 month of age, despite intensive care treatment.

Epilepsy and EEG features. Four out of seven patients suffered from epilepsy (Table 1). Patients #1 and #2 were affected by pharmaco-sensitive epilepsy since the ages of 6 and 3 years, respectively. Seizures were characterized by brief episodes of staring and eyelid myoclonias. EEG recordings displayed diffuse 3.5–5 Hz spike/polyspikes and wave discharges, enhanced by activating procedures, such as hyperventilation and intermittent photic stimulation (IPS). IPS elicited a photoparoxysmal response at frequencies ranging from 6 to 25 Hz (patient #1) and from 6 to 30 Hz (patient #2) (Fig. 1a, b). Patient #3 had the first bilateral tonic-clonic seizure in early adolescence, with good control of the epilepsy after introduction of anti-seizure medications. EEG showed global background slowing. Patient #4 presented with atypical febrile seizures in early infancy and later developed focal non-motor seizures and status epilepticus. Seizure control with anti-seizure medications was achieved at the age of 6 years. From 5 to 8 years of age, he presented with a cognitive regression and language deterioration associated with a remarkable enhancement of the EEG epileptic activity during non-REM sleep compatible with ESES/DEE-SWAS, which later remitted following appropriate treatment, with an amelioration of the cognitive status.

MRI findings. MRI showed malformations of cortical development in four out of six available neuroimaging studies (Fig. 2). In patients #1 (Fig. 2a–c) and #2 (Fig. 2d–f), the MRI disclosed pachygyria, with antero-posterior gradient, sparing the occipital lobes associated with several spotty subcortical white matter abnormalities. Patient #3 (Fig. 2g–i and Supplementary Fig. 2) had focal gray matter heterotopia in the right lateral ventricle and non-specific abnormalities, such as a coarse and small corpus callosum, and enlarged lateral ventricles. In patient #7 (Fig. 2j–l), brain MRI showed cerebral atrophy with enlarged frontal subarachnoidal spaces with septum pellucidum cyst, and brainstem and cerebellum atrophy. In patients #4 and #6, MRI was unremarkable.

Dysmorphisms and other comorbidities:. Description of facial gestalt was available in all patients, and clinical pictures were available in 3/7 patients (#1–3). Patients #1, #2, and #4 were non-dysmorphic. In contrast, patient #3 had upslanting palpebral fissures, medial sparseness of eyebrows, flat nasal bridge and retrognathia (Fig. 3). Twin patients (#5 and #6) showed deep-set hooded eyelids, epicanthal folds, broad nasal tip, thick lips, narrow palate, diastema of teeth, short neck, and unilateral hockey-stick crease. One (#3) out of seven patients had microcephaly, with a range of −3 to −4 SD at last follow-up, and with a progressive course. Patients #5 and #6 had a history of vesicoureteral reflux with recurrent urinary infections, eczema and otitis. The latter was affected by acute meningitis at the age of 4 years. Patient #7 showed multiple contractures of large and small joints, hip dysplasia, arthrogryposis, hirsutism and cryptorchidism. Muscle biopsy revealed wide areas of fibrosis with loss of clear muscle bundles, variability of the size and partial necrosis of muscle fibers (Supplementary Fig. 3). Other features included visual defects (#1) and asthma (#4).

Genetic findings. We identified five variants, not reported previously, four in the following compound heterozygous states—(i) NM_014246.4:c.2935C>T NP_055061.1:p.(Leu979Phe) and c.7456G>C p.(Ala2486Pro); (ii) c.2018T>C p.(Val673Ala) and c.7012G>A p.(Val2338Ile), (iii) c.3601G>A p.(Asp1201Asn) and c.4436T>A p.(Leu1479His), (iv) c.1210G>A (p.Glu404Lys) and c.4415C>G (p.Thr1472Ser) and one homozygous variant c.2935C>T p.(Leu979Phe) in five unrelated families. The variant p.(Leu979Phe) recurred in three Kurdish patients (#1, #2, #4); two of them are siblings and the third patient is unrelated. The other variants were found in one patient each. One variant (c.4436T>A p.(Leu1479His) in patient #5 and #6) occurred de novo. All other variants were inherited from healthy heterozygous parents. All variants were rare (populational allele frequency ≤ 0.0002652) or not observed in the Genome Aggregation Database (gnomAD) representing healthy people (Supplementary Data 1).

Patient #3 carries two additional variants: a de novo (NM_000284.4:c.900-7_932dup, NP_000275.1:p.(?)) duplication in PDHA1 and a paternally inherited (NM_145239.3:c.649dup NP_660282.2:p.(Arg217ProfsTer8)) frameshift variant in PRRT2 variant. Pathogenic PRRT2 variants have been associated with infantile movements disorders, familial infantile convulsions and self-limited epilepsy38, features that are not detected in our patient. Pathogenic PDHA1 variants are causing mild dysmorphism, various neurological symptoms, such as congenital hypotonia, severe encephalopathy, agenesis of the corpus callosum, and diffuse hypomyelination. Furthermore, biochemical abnormalities such as increase of blood alanine, increase of blood and urinary lactic and pyruvic acid, as well as hyperammonemia are often observed in PDHA1 patients39,40. These biochemical abnormalities were not detected in our individual, thus suggesting a limited, if any, role of the PDHA1 variant.

In Silico characterization of CELSR1 biallelic variants
It is acknowledged that most variant predictors lack some in performance. SIFT and CADD has been reported with accuracy of 0.7146 and 0.7198 and a precision of 0.7771 and 0.7453, respectively41. In the same way, Polyphen2 has been reported with a specificity of 0.73 and a sensitivity of ~0.8742. Thus, we decided to comprehend the structural consequences of the ascertained genetic variants, by using 3D structural models based on sequence homology to known protein crystal structures (Fig. 4 and Supplementary Fig. 4).

The crystal structure of the extracellular cadherin domains 1 to 4 of CELSR1 is reported as PDB no. 8D40. The position of the genetic variant p.(Glu404Lys) is situated in CELSR1 cadherin domain 2 in the loop between strands E and D. Glu404 stabilizes the loop forming polar contacts to Tyr390 (Fig. 4a). The substitution Glu404Lys destabilizes the E-D-loop destabilizing the cadherin repeat.

Embedded in the beta strand A of cadherin domain 4, Val673 displays complete conservation between CELSR1, 2 and 3, and a high conservation throughout the Cadherin Major Branch (CMB) (Supplementary Fig. 4), almost only being replaced by other amino acids with hydrophobic side chains. In the crystal structure of human CELSR1 extracellular cadherin domain 4–7, PDB no. 7SZ8. Val673 is positioned in strand B establish backbone hydrogen bonds to Phe655 on strand E (Fig. 4b). The substitution Val673Ala destabilizes the E-D-loop and thereby the cadherin domain. The Ser741Ile substitution in CELSR3 corresponds to the Ser672Ile substitution in CELSR1 which previously has been reported as pathogenic causing tetralogy of Fallot25.

The residue Leu979 is situated in the linker between cadherin domains 7 and 8. The position is illustrated in a crystal structure based on Homo Sapiens Protocadherin GammaB4, PDB no. 6E6B ref. 43 (HHpred Probability: 99.74%, E-value: 6,8e-11, PHYRE2 confidence 100, 00%) (Fig. 4c). Polar interactions are shown. Leu979 displays interaction with Asp972. Substituting Leu979 with phenylalanine will cause steric interference with Asn886 and additionally destroy the bond to Asp972, both involved in Ca2+ binding. The result will be destabilization of the essential extracellular Ca2+-binding domain of the cadherin repeat, leading to a dysfunctional protein structure.

The position of Asp1201 is deduced using the crystal structure of Sus Scrofa Protocadherin-15, PDB no. 6BXZ ref. 44 (HHpred probability: 98.08%, E-value: 0.0067) (Fig. 4d) and is part of the ninth cadherin domain of CELSR1. Asp1201 is part of the Ca2+-binding loop between strands B and C, stabilizing the loop forming backbone hydrogen bonds to Leu1203, Thr1204, and Asn1205. The substitution Asp1201Asn will result in steric clashes with Asn1205.

The crystal structure of Bos Taurus Neurexin-1-alpha PDB no. 3POY ref. 45 (HHpred probability 99.58%, E-value: 4.4e-13, PHYRE2 confidence 99.9%) served as a model for the laminin G-like 1 domain harboring Thr1472 (Fig. 4e) and Leu1479 (Fig. 4f). Thr1472 forms backbone hydrogen bonds to Gly1525 and hydroxyl bond to Glu1474 and Arg1838. The p.(Thr1472Ser) substitution will destabilize the loop between strand C and D of the globular structure. Leu1479 is positioned in strand D forming hydrogen bonds to Leu1494 and Gly1268 in KL-loop harboring the highly conserved cysteine disulfide bridge between Cys1840 and Cys1870. The p.(Leu1479His) substitution will result in steric interference with Thr1472 and Gly1477. The GAIN-GPS has been modeled using the crystal structure PDB no. 4DLQ ref. 46 of Rattus Norvegicus CIRL 1/Latrophilin 1 (CL1) (HHpred Probability: 99.2%, E-value: 1e-9, Phyre confidence 99, 95%, intensive mode). The GAIN (G—protein-coupled receptor (GPCR) autoproteolysis-inducing domain) domain of CELSR1 is followed by the GPS domain. Val2338 is situated in the olfactomedin part of the GAIN domain (Fig. 4g) and is part of the GAIN domain binding pocket for the tethered agonist. Situated in sheet 11, Val2338 forms two polar bonds to Phe2253 in sheet 8. Introduction of the p.(Val2338Leu) substitution will cause a change in distance between these two b-sheets (11 and 8) hypothesized to cause a conformational change destabilizing the GPS-autoproteolytic-GAIN domain that then is predicted to decrease or abolish the essential autoproteolysis function at the site between sheet 19 and 20.

For the transmembrane region of CELSR1, a crystal structure based on Homo Sapiens adhesion G protein-coupled receptor L3 (AGRL3), PDB 7SF7 ref. 47 (HHpred Probability: 99, 93%, E-value: 2, 3e-22, SWISS-MODEL Ramachandran Favored 92, 86%, PHYRE2 Confidence 99, 9%, intensive mode) was used (Fig. 4h). This region consists of seven transmembrane alpha helixes (TM1–TM7). Ala2486 is situated in the alpha helix constituting TM1. Substituting Ala2486 with a proline will cause a kink and is not compatible with alpha helical structure. The p.(Ala2486Val) substitution is seen in GnomAD. However, alanine and valine are two amino acids with very similar structures and size; hence, this substitution is in contrast to the p.(Ala2486Pro) substitution predicted to preserve the alpha helical structure.

In Supplementary Table 1, the quality parameters of the templates used are listed. Taken together, the above predicted 3D structures present with a very high structural confidence, supporting the strength of the predicted functional consequence of the genetic variants. The recently published structure of CELSR1 is in agreement with our data48. PyMol sessions are available as Supplementary Data 2.

The phenotype of Celsr1 KO mice recaps the human findings
We have previously reported that Celsr1 KO mice exhibit defective embryonic neurogenesis and cilia polarization that result in cortical hypoplasia, enlarged lateral ventricles, learning and memory deficits, and autistic-like behavior30,36. Here, we focused on the postnatal brain. We examined coronal sections from Celsr1 KO mice (n=17 mice from 33 litters), heterozygotes (n=119), and control littermates (n=61) following Cresyl Violet (Nissl bodies), and Luxol Fast Blue (myelin) staining. Heterozygous mice were indistinguishable from controls. Celsr1 KO mice exhibited agenesis of the caudal corpus callosum (Fig. 5a, b). The phenotype was fully penetrant and background independent, as backcrossing on C57Bl/6 or CD1 (eight generations) did not affect the phenotype. The hippocampal commissure was also absent in the KO mice. Tag1 immunostaining at postnatal day (P) 0, showed that pioneer callosal axons failed to cross the midline and run along the rostral-caudal axis, forming so-called Probst bundles (Fig. 5c). Glial cells of the indusium griseum were absent in KO mice. The glial wedge that behaves as a repulsive cue toward extending axons49, although present, showed a wider interhemispheric spacing (Fig. 5c).

To further characterize the callosal axon defects, we performed axonal tracing using DiI, which confirmed the absence of crossing callosal axons and emphasized their rostrocaudal as well as ventral trajectories (Fig. 5d).

The Celsr1 KO mice also displayed periventricular heterotopia, particularly around the subventricular zone (Fig. 6a–d). Heterotopias are usually due to fate determination and/or neuronal migration defects. We have previously reported that during embryogenesis, neural progenitors undergo more proliferative divisions at the expense of neuron production, leading to local accumulation of cells and widening of the ventricular/subventricular zones (Fig. 6b)36. To assess radial migration, we injected pregnant dames intraperitoneally with bromodeoxyuridine (BrdU) at embryogenic day (E) 14.5 and analyzed the brain at postnatal day (P) 0. Like in control mice, BrdU- and Cux1-positive neurons were seen in the outer tier of cortical ribbon in Celsr1 KO, suggesting that radial migration, layering, and inside-out gradient of cortical maturation were not dramatically affected by the lack of CELSR1 (Fig. 6e, f). However, the density of BrdU-positive cells was significantly higher in the mutant ventricular zone (Fig. 6f, g; Source Data File).

In the postnatal brain, Celsr1 mRNA was expressed in the subventricular zone and the rostral migratory stream (RMS), and its loss led to a marked atrophy of olfactory bulbs (OB) in adult mice (Fig. 7a–c). We used an antibody against Doublecortin (Dcx), which specifically labels neuroblasts and delineates the RMS. In control mice, Dcx-positive cells were located in OB and along the RMS. These cells accumulated around the ventricles, particularly in the SVZ, and in the proximal RMS in Celsr1 KO (Fig. 7d, e). Dcx immunoreactivity was also detected ectopically in the frontal cortex. To further characterize the tangential migration of neuroblasts, we injected adult mice intraperitoneally with BrdU for 3 consecutive days and analyzed the distribution of BrdU-positive cells 9 days after the last injection (~5 days are necessary for adult neural stem cells to differentiate into neuroblasts and migrate from SVZ to OB). Immunostaining with anti-BrdU antibodies showed that the number of BrdU-positive cells was greater in the Celsr1 KO SVZ compared to controls (Fig. 7f–k; Source Data File). These results, together with Dcx immunostaining, suggest that neuroblast migration is disrupted in Celsr1 KO mice. Instead of moving rostrally toward OB, they accumulated in the periventricular areas, leading to a progressive depletion of olfactory interneurons and hypotrophy of OB.

Table 2 summarizes the brain malformations in KO mice in comparison with our patients. A small proportion (20%) of Celsr1 KO mice exhibited spontaneous epileptic seizures starting from the third postnatal week (Supplementary Movie 1). We evaluated the susceptibility of KO mice to pentylenetetrazole (PTZ)-induced seizures in comparison with control littermates. Age- and sex-matched animals of different ages (2–12 months, n=10 per genotype) were injected with 35 mg/kg of PTZ and visually monitored for 30 min. Control mice displayed neither behavioral nor immobilization disturbances. In sharp contrast, young adult KO mice (≤4 months, n=4) had generalized seizures (Supplementary Movie 2), whereas mature KO mice (>4 months, n=6) displayed continuous whole-body myoclonus. Uncropped and unprocessed data of Fig. 5–7 are available as Supplementary Fig. 5

Methods
Patient’s recruitment
Patients were collected through data sharing among Epilepsy and Genetic Centers in Europe and the United States. Data were included and categorized in a common database stored at the Danish Epilepsy Centre. Grouping of clinical data for this series was facilitated by GeneMatcher63. All patients were clinically evaluated by neuropediatricians, neurologists and clinical geneticists at their respective tertiary healthcare centers. Patients underwent clinically indicated evaluations, including detailed clinical history and physical examinations, genetic testing, EEG, and MRI. Available EEG recordings (either during wakefulness and sleep) and brain MRIs were obtained and reviewed for all patients. Brain MRIs were reviewed and visually analyzed by the same neuroradiologist (CGM) according to standard clinical practice. Various MRI protocols were used, which were of varying field strengths, slice thicknesses, and image qualities (3T DICOM format to 6-mm slice thickness; hard copy film photos at 1.5T). Seizures and, where possible, epilepsy syndromes were classified according to the International League Against Epilepsy (ILAE) classification proposals7,64. Inconsistencies and discrepancies between reported clinical and diagnostic data were resolved by seeking further information from referring physicians.

Molecular genetic analysis
All CELSR1 variants were provided through diagnostic laboratories where they have been identified as part of standard clinical formal diagnostic work-up in patients with epilepsy, cognitive impairment, and/or cortical malformations. We received the results as written reports of the genetic analyses, including the precise variant positions and their ACMG classifications. We did not review the raw exome or Sanger sequencing data; only information regarding the specific variants was provided. The pathogenicity of the genetic variants was classified according to ACMG guidelines65. Only class III-V variants are reported here. Class III variants are defined as “variants of unknown clinical significance”, class IV as “likely pathogenic” and class V as “pathogenic” variants. Included in this evaluation were database searches in NCBI PubMed, Human Gene Mutation Database (HGMD 2024.1), the Genome Aggregation Database (gnomAD, release 4.1.0), Exome Aggregation Consortium (ExAC), dbSNP (155), ClinVar, and PER viewer [PER.BROADINSTITUTE.ORG/]. To fulfill our filter criteria, the populational allele frequency of the variants should be lower than 1% based on the frequencies given in the current version of public variant databases gnomAD and ExAC, and the variants should be absent in the homozygous state from gnomAD and ExAC. In silico evaluation using SIFT, PolyPhen2, MutationTaster, and CADD, was performed to predict the effect of variants on protein function and the evolutionary conservation of the amino acid at the specific position of interest.

Bioinformatic analysis and protein modeling
The SMART SERVER [SMART.EMBL-HEIDELBERG.DE/] was used for assessment of the functional domains of CELSR1. The structure of each functional domain was obtained individually by performing analysis for structural homology using HHPRED66 [TOOLKIT.TUEBINGEN.MPG.DE], PHYRE267 [WWW.SBG.BIO.IC.AC.UK/PHYRE2], I-TASSER68 [ZHANGLAB.CCMB.MED.UMICH.EDU/I-TASSER/], or RAPTORX69 [RAPTORX.UCHICAGO.EDU/], SWISS-MODEL70 [SWISSMODEL.EXPASY.ORG/INTERACTIVE]. When two or more modelers predicted a model, this was selected based on the highest template-model score, if additionally supported by sequence similarity based on multiple sequence alignment (MSA) with an identity above 30% and functional homology of the template. Templates with a resolution below 4.5 Å and an R value below 0.25 was selected. For all tools, standard settings were used except for Phyre2, for which intensive mode was utilized. Models were retrieved from RCSB PDB [WWW.RCSB.ORG]. Retrieved model sequences were aligned to CELSR1 RefSeq NP_055061.1 using MUSCLE.

Mutant mice
The Celsr1 KO mice were described in Ravni et al.31 and Boucherie et al.36. The experiments were carried out on both males and females without any distinction of the gender.

Immunohistochemistry
We used 12-μm-thick cryosections and 50-μm-thick vibratome slices from postnatal brains fixed with 4% paraformaldehyde. Sections were blocked in PBS supplemented with 0.5% Triton X-100 and 3% bovine serum albumin and incubated with the following primary antibodies: rabbit anti-GFAP (Millipore, Cat. N° AB5804, 1/1000), mouse anti-Tag1 (Developmental Studies Hybridoma Bank, Cat. N° 4D7, 1/40), rat anti-BrdU (Serotec Cat. N° MCA2060GA, 1/100), Rabbit anti-Cux1 (Proteintech Cat. N° 11733-1-AP, 1/1000), rabbit anti-Doublecortin (Dcx, Cell signaling Cat. N° 4604, 1/800). Different Alexa Fluor-conjugated secondary antibodies (Invitrogen, 1:800) were used. After immunohistochemistry, the sections were incubated with DAPI (Sigma D9564 100μM) for 5 min and mounted with Mowiol.

Histology
For histological examination, 8-μm-thick paraffin sections were deparaffinized, hydrated, and incubated with Cresyl Echt Violet (0.1% in distilled water) for 5 min to assess cell density and cortical architectonics, or with Luxol Fast Blue solution (0.1% in 95% ethanol with 0.5% acetic acid) for 24 h at 60 °C to assess the myelin content and axonal tract integrity. The sections were rinsed in deionized water and differentiated in 70% ethanol (Cresyl violet), or 0.05% lithium carbonate solution, followed by 70% ethanol (Luxol Fast Blue). Finally, the sections were dehydrated in an ethanol series, cleared in xylene, and mounted with DPX.

DiI tracing
DiI crystals (D3911, Molecular Probes) were implanted in target areas (cortical layers 2–3) of PFA-fixed brains using tungsten needles. Blocks were incubated in the dark at 37 °C for 4 weeks and sectioned using a vibratome. About 150-μm-thick sections were counterstained with DAPI (0.25 μg/ml) were mounted with Mowiol medium, and examined under epifluorescence microscopy with a rhodamine filter.

In situ hybridization
Total RNA was extracted from normal newborn mouse brains using a RNeasy kit (QIAGEN) according to the manufacturer’s instructions. To produce the riboprobe, the first strand cDNA was synthesized from 10 μg of total RNA using Superscript III reverse transcriptase (Invitrogen). cDNA of the Celsr1 genes were amplified by RT-PCR using the following primers: forward: 5’-CCACCCCTGAAATCTGACC-3’, reverse 5’-CACTCTGTATCCTTGCCATCC-3’. In situ hybridization (ISH) was carried out with [alpha-P33]UTP-radiolabelled riboprobe. Briefly, cryostat sections were treated with 1 lg⁄mL proteinase K in 0.1 M Tris, pH 8–10 mm EDTA, rinsed in DEPC-treated water, and acetylated for 10 min in 0.25 M acetic anhydride and 0.1 M triethanolamine solution. Slides were incubated overnight at 55–58 °C in a humid chamber with denatured probes (1–5.106 dpm⁄slide) in hybridization solution [50% formamide, 10% dextran sulfate, 0.3 M NaCl, 20 mM Tris–HCl, pH 7.5, 5 mM EDTA, Denhardt’s solution (1x), 0.6 mg⁄mL yeast tRNA, and 0.1% sodium dodecyl sulfate (SDS)]. Following hybridization, slides were washed for 30 min at 65 °C in 50% formamide, 2x standard sodium citrate (SSC) solution, rinsed in 2x SSC, and treated for 1 h at 37 °C with 20 lg⁄mL RNAse A in NTE buffer (0.5 mM NaCl, 10 mM Tris–HCl, pH 7.5, and 5 mM EDTA). Slides were finally washed in 2x SSC at 60 °C for 1 h and in 0.2x SSC at 65–68 °C for 2 h to achieve high stringency and exposed to Kodak BIOMAX-maximum resolution films.

BrdU injection and quantification
To label dividing cells, BrdU (50 mg/kg body weight) was injected into pregnant females at embryonic day 14.5 (E14.5), and brains were collected at birth (P0). For postnatal neurogenesis experiments, Celsr1 KO and control littermates (3 months old mice), were subjected to intraperitoneal injection of BrdU for three consecutive days and euthanized 9 days after the last injection. For BrdU staining, cryosections and vibratome sections were pretreated with 2N HCL for 30 min, followed by a 10 min incubation with borate buffer and immunodetection using primary antibody mouse rat anti-BrdU (Serotec Cat. N° MCA2060GA, 1/100). For BrdU quantification in the SVZ of adult mice, four sections were selected at rostral and caudal levels for each animal (n=3 animals per genotype). BrdU-positive cells were quantified manually using ImageJ software in an area of 600,000 μm2, and values were normalized per mm2. Statistical analysis and graphs were constructed using Prism 9 (GraphPad, San Diego, CA, USA) software. The exact sample size is specified in the figure legends. Error bars represent the standard error of the mean (SEM). We performed a Shapiro–Wilk test to evaluate the distribution of the data. We then used a two-tailed Student’s t-test since the data followed a normal distribution (n.s. not significant, *p<0.05, **p<0.01, and ***p<0.001).

Pentylenetetrazole (PTZ)-mediated kindling
For PTZ-induced sensitization, Celsr1 KO and control littermates (n=10 per genotype) were injected intraperitoneally with 35 mg/kg of PTZ and visually monitored for 30 min. Their behavior was scored and grouped into seven categories: (0) normal behavior; (1) immobilization, lying on belly; (2) head nodding, facial, forelimb, or hindlimb myoclonus; (3) continuous whole-body myoclonus, myoclonic jerks, tail held up stiffly; (4) rearing, tonic seizure, falling down on its side; (5) tonic-clonic seizure, falling down on the back, wild rushing and jumping; and (6) death.

Microscopic imaging analyses
High-resolution images (1024×1024 pixels) were captured with a digital camera coupled with an inverted Zeiss Axio Observer microscope or in a Laser Scanning confocal microscope (Olympus Fluoview FV1000) using the ZEN microscopy software and the FluoView FV1000 Software, respectively. Color images were obtained using optical microscopy (ZEISS AxiosKop). Images were processed using ImageJ software, and figures were prepared using Adobe Photoshop and Adobe Illustrator CC 2019, a software package. All the figures containing microscopy images were prepared following the image integrity guidelines from Nature Portfolio.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Bonardi and collaborators report on a novel developmental and epileptic encephalopathy caused by biallelic variants in\nCELSR1. In the manuscript, they describe the clinical phenotype of eight patients carrying six homozygous or compound\nheterozygous variants in CELSR1, assess the potential pathogenicity of the variants through in silico 3D modelling and\nprediction tools, and carry out an extensive analysis of the brain of Celsr1 KO mice.\nMajor points\nAs also reported by the authors, heterozygous LOF mutations in CELSR1 have been associated with lymphoaedema and\nOMIM describes the gene as causative of lymphatic malformation 9 (OMIM 619319). In the patients series described in the\nmanuscript, lymphoaedema has been reported only in the mother of Patient 6. The reason why none of the patients carrying\ncompound heterozygous or homozygous variants in CELSR1 exhibit lymphatic anomalies should be hypothesized in the\ndiscussion section.\nParagraph “2.1.4 MRI findings”: The paragraph should be reorganized citing the findings observed in the patients according\nto how they are presented in Figure 2 (i.e. Patient 1, 2, 3, 4, and 8).\nParagraph “2.1.6 Genetic findings”: Any additional finding identified through WES should be disclosed (e.g. in a\nsupplementary table).\nParagraph “2.2 Molecular characterization of rare CELSR1 biallelic variants”: Although the authors provide a detailed\ninterpretation of the position of the mutated amino acids within the protein in the paragraph, for some variants (Glu404Lys,\nVal673Ala, and Val2338Ile) they do not indicate the expected effect of the substitution. For Glu404Lys and Val2338Ile this\nprediction is particularly important, as they have been classified as benign by in silico predictors.\nFigure 2: Arrows are not clearly visible in and their color should be changed to white or black.\nImage 4: The quality of Image 4 is poor. The name of amino acids and some drawings are unreadable, at least in the version\nof the manuscript submitted for revision.\nFigure 5: The authors do not provide explanations of the meaning of what is shown in panel C.\nFigure 6C: Scale bar should be moved in another place, as it is located on the periventricular zone of the brain and does not\nallow a proper comparison between WT and mutant.\nFigure 7 legend: The authors stated that \"the number of BrdU-positive cells was greater in the Celsr1 KO SVZ compared to\ncontrols\". However, they do not provide cells quantification and their assumption is probably based on the higher amount of\nfluorescence observed in mutant brains with respect to WT. The authors should provide cells quantification or change the\nsentence accordingly.\nMinor points\nLine 227: for the sake of clarity, “two brothers of related parents” should be changed to “two sons of related parents”.\nLine 247 “The EEG showed…”: The sentence should be reworded.\nLine 293 “2.2 Molecular characterization of rare CELSR1 biallelic variants”: the title should be changed to “in silico 3D\ncharacterization of rare CELSR1 biallelic variants” or somewhat similar, as the authors did not perform a real molecular\ncharacterization of the variants.\nLine 305 “Based on the crystal structure…”: The sentence should be reworded.\nLine 412: \"…been associated brain malformations…\" should be changed to \"…been associated with brain malformations…\n\".\nLine 827: In the figure, we can only appreciate microcephaly, thus \"progressive\" should be removed.\nIn the whole manuscript, panels are indicated in uppercase in the figures and in lowercase in the text and legends.\nSupplementary material, Line 17: “mental retardation” should be changed to “intellectual disability”.", "reviewer_2": "Authors report the clinical features of 8 patients from 6 families with biallelic CELSR1 missense variants. They also\nperformed protein modeling and KO mice studies of the variants. They identified similar findings between human patients\nand model mice, such as microcephaly, dilated lateral ventricles abnormal corpus callosum, and periventricular heterotopia.\nWhile CELSR1 variants have been reported in patients with lymphedema, neural tube defects, epilepsy, and cardiac\nanomalies, their findings, brain malformations in particular, would expand phenotypes of the\nCELSR1 variants and suggest genotype-phenotype correlation. However, the manuscript needs to be restructured before publication.\nThe descriptions of the Introduction and Discussion are lengthy and too scholastic. They should focus on the original\nfindings of this study.\nThey insist that the brain MRI of Patients #1 and #2 showed “severe” and “diffuse” pachygyria, however, their occipital lobes\nappear to be spared in Fig.2A-F. They need to define the meaning of “severe” and mark the pachygyria in the occipital\nregion.\nThey mention, “diffuse” subcortical white matter abnormalities, however, Fig.2A and D do not show diffuse changes. They\nlook spotty. They also consider the white matter abnormalities as “dysmyelination” described in Supplementary material,\nhowever they might be gliosis. How did they discriminate gliotic changes from the abnormal intensities in the white matter?\nIt's hard to see periventricular heterotopia in Fig.2J. Coronal views with a magnified inset would be helpful to show them.\nAs for the patients’ faces, both patients appear to show thin upper lip (vermilion) and bulbous nose in Fig.3. Please recheck\nthem.\nPatients #1 and #2 showed photosensitivity on EEG. It would be helpful to mention the range of photic stimulation frequency\nwhen photoparoxysmal response occurred. Patient #1 showed a paroxysmal discharge triggered by eye-closure. It seems to\nbe fixation-off sensitivity. If so, it would be better to briefly mention it.\nPatient #8 appears to show dystrophic changes in muscle biopsy. It would be helpful to show some figures of muscle\npathology and discuss the relationship with the CELSR1 variants.\nIt’s hard to see Figure 4 due to low resolution. Please fix it.\nIt would be better to clarify and discuss if and why the radial migration was normal and the tangential migration was\nabnormal in Celsr1 KO mice.\nWhen did Celsr1 KO mice start to exhibit spontaneous seizures?\nPhenotype differences between the previous reports of heterozygous variants and this study of biallelic variants were\nshown, however, they insist no phenotype-genotype correlations have been identified in the conclusion. They should\nreconsider it.\nMinor points\nIn the figure legends, IPS and ESES would not need to abbreviate themselves because they appear once in the legends\nFig.2H is upside-down.\nCase reports in the supplementary material should be described in the past tense.\nThe term, “mental retardation”, should be changed to “intellectual disability”.\nDelete the year when WES was performed in each patient. It is unnecessary to enhance medical accuracy for this study.\nESES usually means “Electrical” status epilepticus in sleep [Specchio N, et al. Epilepsia 2022;63:1398-1442]. Not\n“Encephalopathy related to”.\nCPK should be CK in formal and would be better spelled out.\nIn Supplementary Table 1, they classify the variants as III or IV, possibly based on the ACMG guideline. The original ACMG\nguideline published in 2015 mentions i) pathogenic to v) benign. Do they mean IV is likely benign?", "reviewer_3": "The authors report the phenotype of 8 individuals from 6 unrelated families with bi-allelic variants in CELSR1. Pathogenicity\nand functional effects of the variants were inferred by bioinformatic analysis, protein modelling and prediction tools. Further\nsupport for the role of CELSR1 in neurodevelopment was provided by description of the clinical phenotype and analysis of\nthe adult brain of Celsr1 KO mice.\nThis report significantly adds to the current knowledge on brain development and deserves publication.\nThere is one major point I would like the authors to address prior to publication, which is statement that bi-allelic variants in\nCELSR1 are a cause of Developmental and Epileptic Encephalopathy (DEE).\nIn the introduction, the authors include the definition of DEE as follows “Developmental and Epileptic\nEncephalopathies (DEE) form a group of severe childhood onset disorders characterized by neurodevelopmental regression or plateauing\noften associated with early onset refractory epileptic seizures. Epileptic activity “per se” can contribute to severe cognitive\nand behavioral impairment, above what is expected from the underlying pathology, and the developmental\nconsequences may improve with the amelioration of the epileptiform activity.” In the CELSR1 cohort reported here, only 1 out of 8\nindividuals (patient #5) showed a neurodevelopmental regression in the context of a DEE-SWAS. Only 2 additional\nindividuals had epilepsy which did respond to anti-seizure medication. I therefore disagree to classify the phenotype\nassociated with bi-allelic variants as an example of a DEE based on the information available to date. In line with this view, I\nwould suggest to adapt the introduction with less focus on DEE and rephrase the statements linked to DEE throughout the\nrest of the manuscript, including the abstract.\nI have a few minor suggestions that might improve the manuscript.\nI would propose to include a sentence on the high degree of phenotypic variability in this cohort in the abstract and to\nhighlight this a bit more in the discussion. Brain abnormalities were indeed present in most individuals but the type of\nabnormality varied considerable between individuals. The degree of developmental impairment was also highly variable\nranging from one child dying in infancy to two adolescents with much milder phenotypes. Epilepsy was present in only 3 out\nof 8, again with variable seizure types and response to treatment.\nAbstract: include white matter changes as part of the brain abnormalities.\nAbstract: If word count allows I would propose to include the increased susceptibility to seizures in the Celsr1 KO mice since\nthis provides an additional link to part of the human phenotype.\nIntroduction: “CELSR1 is key to ependymal cilia polarization and ciliary beats and its loss severely disrupts circulation of the\ncerebrospinal fluid and results in hydrocephalus. Variants in the CELSR1 gene have been linked to congenital defects in the\nvestibulocochlear system, heart, kidney, and lung.” Based on these findings, could the CELSR1-related phenotype be\nclassified as a ciliopathy? Arguments in favour/against?\nIntroduction: “In the developing rhombencephalon, CELSR1 specifies the directionality of tangential migration of facial nerve\nmotor neurons. CELSR1 is also key to ependymal cilia polarization and ciliary beats and its loss severely disrupts\ncirculation of the cerebrospinal fluid and results in hydrocephalus.” It would be helpful if you could mention if these\nconclusions were based on work in mice or other models.\nResults: Epilepsy phenotype – replace ESES by D/EE-SWAS as per latest ILAE report? Idem for Table and Case\nDescription in Supplementary Materials.\nDiscussion: “Pachygyria is included in the larger group of lissencephalic syndromes, and it is characterized by decreased\ncortical convolutions, cortical thickening, and a smooth cerebral surface.” More recent references to the classification of\nlissencephaly include PMID 28440899 and PMID 29671837.\nDiscussion: “Pathogenic variants in ACTB and ACTG1 genes have also been associated with pachygyria.” Not relevant to\nrefer to only these two genes. not relevant to highlight only these two genes. Pachygyria a>p can be seen with variants in\nDCX, ACTB, DYNC1H1, ACTG1, KIF5C, VLDLR, CRADD, or ARX (in that case with ACC). Reference PMID: 32895508.\nDiscussion: “PNH consists of nodules of grey matter located along the lateral ventricles with a total failure of migration of\nsome neurons.” More accurate reference includes PMID: 32895508.\nDiscussion: Three individuals has abnormal corpus callosum. It is unclear from the images and table which patients are\nconcerned. Patient 4 has a convincingly short and thick corpus callosum. Patient 3 is reported to have a course small corpus\ncallosum in the Table but this is not clear in Figure 3I. There is no third individual mentioned with callosal abnormalities in\nthe Table.\nFigure 2\nFigure 2C: Cerebellar vermis hypoplasia?\nFigure 2G-I: patient #3 – Since I do not have access to the entire MRI it is difficult to judge the cortical pattern but based on\nFig2H I have the impression that the gyration is not normal either in this individual.\nFigure 2J: The periventricular nodular heterotopia are unclear on this image. Would it be an option to provide an image in\nthe axial plane to illustrate the PNH?\nFigure 2M-0: patient #8: cortex abnormal – double check with radiologist?\nTable 1: head circumference now either by centile or standard deviation. Since the definition of micro/megalencephaly is\nbased on SD, it would be preferred to have all SD for all head circumference measures.\nSupplementary material:\nPatient #1: please replace ‘mental retardation’ by ‘intellectual disability’\nPatient #6: autism spectrum disorder, level 2 – unclear what ‘level 2’ stands for.\nPatient #8: “Later a trio WES and whole genome sequencing (WGS) were performed and disclosed two missense variants,\nc.1210G>A (p.Glu404Lys) and c.4415C>G, (p.Thr1472Ser) in CELSR1 with maternal and paternal inheritance,\nrespectively.”", "reviewer_4": "The description of the structural prediction is insufficient and the validity is unclear.\nFirst, it is necessary to properly describe how multiple prediction tools are used, along with the reasons. It is also necessary\nto describe all of the options used to predict each tool (for example, the prediction results of Phyre2 differ between Normal\nmode and intensive mode).\nIn homology modeling, the reliability of the prediction varies greatly between the regions covered by the template and those\noutside the template. Therefore, it is necessary to provide information about the template and sequence alignment used for\nall predictions. E-values are calculated from the entire sequence alignment and do not guarantee local quality, so they are\ninsufficient for the current discussion.\nIt is also necessary to discuss the quality of the template structure. For example, PDBID:6C13 is an EM structure with a\nresolution of 11.3 angstroms, and it is considered to be an inappropriate template for the current discussion.", "reviewer_5": "This study describes the phenotype of eight individuals with biallelic variants of CELSR1, presenting a novel epileptic\nencephalopathy characterized by brain malformations, neurodevelopmental delay, behavioral disorders, and epilepsy.\nWhole exome sequencing revealed compound heterozygous and homozygous variants. Analysis of Celsr1 mutant mice\nshowed phenotypes similar to those found in humans, such as partial agenesis of the corpus callosum, periventricular\nheterotopia, and enlarged lateral ventricles, highlighting the critical role of CELSR1 in human neurodevelopment. While not\nfinding an exact parallelism between mouse and human phenotypes, mutant mice for this gene could serve as a model for\nstudying this disorder. Interestingly, the authors suggest that potential treatment with retinoic acid administered during\npregnancy could help alleviate this condition in the future. It is an intriguing study but there are several issues that need to be\naddressed before publication:\n1. Since most patients are heterozygous, it would be interesting to know to what extent heterozygous mice exhibit similar\nphenotypes.\n2. Line 361: Although the authors state that they focus on the analysis of adult brain, many analyses are actually conducted\non newborn animals.\n3. Why do they analyze P0 mice in Figure 5 and P18 mice in Figure 6? It would be desirable to analyze both ages in both\ncases.\n4. In panels with high-magnification pictures, it is challenging to identify the specific brain area. Including drawings or lower-\nmagnification photographs indicating the specific area shown in the high-magnification photo would be helpful.\n5. Figure 6: What is the conclusion of the BrdU injection experiments? Is there increased proliferation in mutants? These\nresults should be quantified. Additionally, the control photo does not clearly show BrdU-positive cells because the ventricle\nedge is not visible.\n6. Figure 7: i/The expression of Celsr1 in the olfactory bulb is intriguing, but as it is a null mouse line, correlating this\nexpression with observed defects may not be valid. Defects could be due to developmental issues. ii/Including drawings or\nlower-magnification sections indicating the photographed area in panels D and E would be helpful. iii/ Adding labels with\nthe names of the structures shown would also aid comprehension.\n7. Line 396: The word \"show\" in \"Dcx immunostaining show that neuroblast migration is disrupted...\" should be replaced\nwith \"suggest.\"\n8. The epilepsy results in the Supplementary Table should not be supplementary data but included in the main text, as they\nrepresent a phenotype mimicking that of humans"}, "author_response": "August 13th, 2024\nPoint-by-point response to referees\n“Biallelic variants in CELSR1 cause brain malformations, neurodevelopmental delay, and\nepilepsy in humans”\nWe thank the reviewers for their constructive comments and feed-backs that have contributed to\nimprove significantly our paper. In this revised manuscript, we have tried to comply with all the\nreviewer’s comments and suggestions, and we have incorporated all the changes in the text,\nhighlighted them by using a colored text. The responses to the concerns raised by the reviewer are\nalso reported below in a point-by-point fashion.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nBonardi and collaborators report on a novel developmental and epileptic encephalopathy caused\nby biallelic variants in CELSR1. In the manuscript, they describe the clinical phenotype of eight\npatients carrying six homozygous or compound heterozygous variants in CELSR1, assess the\npotential pathogenicity of the variants through in silico 3D modelling and prediction tools, and\ncarry out an extensive analysis of the brain of Celsr1 KO mice.\nMajor points\nAs also reported by the authors, heterozygous LOF mutations in CELSR1 have been associated\nwith lymphoedema and OMIM describes the gene as causative of lymphatic malformation 9\n(OMIM 619319). In the patients series described in the manuscript, lymphoedema has been\nreported only in the mother of Patient 6. The reason why none of the patients carrying compound\nheterozygous or homozygous variants in CELSR1 exhibit lymphatic anomalies should be\nhypothesized in the discussion section.\nreported in patients with heterozygous protein truncating or splice-site variants in CELSR1, which\ncould indicate that haploinsufficiency is the primary disease mechanism in CELSR1-related\nlymphedema. (Erickson, R. P., Lai, L.-W., Mustacich, D. J., Bernas, M. J., Kuo, P. H., Witte, M. H.\nSex-limited penetrance of lymphedema to females with CELSR1 haploinsufficiency: a second\nfamily. Clin. Genet. 96: 478-482, 2019; Gonzalez-Garay, M. L., Aldrich, M. B., Rasmussen, J. C.,\nGuilliod, R., Lapinski, P. E., King, P. D., Sevick-Muraca, E. M. A novel mutation in CELSR1 is\nassociated with hereditary lymphedema. Vasc. Cell 8: 1, 2016.; Maltese, P. E., Michelini, S., Ricci,\nM., Maitz, S., Fiorentino, A., Serrani, R., Lazzerotti, A., Bruson, A., Paolacci, S., Benedetti, S.,\nBertelli, M. Increasing evidence of hereditary lymphedema caused by CELSR1 loss-of-function\nvariants. Am. J. Med. Genet. 179A: 1718-1724, 2019). All patients reported in this study harbor\nbiallelic missense variants, and this could be the reason for the different, mainly\nneurodevelopmental, phenotype (page 25, lines 700-704).\nParagraph “2.1.4 MRI findings”: The paragraph should be reorganized citing the findings observed\nin the patients according to how they are presented in Figure 2 (i.e. Patient 1, 2, 3, 4, and 8).\nAuthor’s reply: we thank the reviewer for this suggestion, and we have modified the text\naccordingly (page 12, lines 338-348).\nParagraph “2.1.6 Genetic findings”: Any additional finding identified through WES should be\ndisclosed (e.g. in a supplementary table).\nAuthor’s reply: In patient #3 WES has revealed two additional variants: a de novo\nPDHA1 variant\nand a paternally inherited PRRT2 variant. These additional findings have been reported in the\nsupplementary material in the clinical description of each patient (supplementary material) and\nalso in the 2.1.6 paragraph (page 14, lines 385-396).\nWe have added these sentences to the text: “Patient #3 carries two additional variants: a de novo\nPDHA1 and a paternally inherited PRRT2 variant. PRRT2 variants have been associated with\ninfantile movements disorders and self-limited epilepsy, features that are not detected in our\npatient. Pathogenic PDHA1 variants are causing mild dysmorphism, various neurological\nsymptoms, such as congenital hypotonia, severe encephalopathy, agenesis of the corpus callosum\nand diffuse hypomyelination. Furthermore, biochemical abnormalities such as increase of alanine,\nlactic and pyruvic acid and, hyperammonemia are often observed. These biochemical\nabnormalities were not detected in our individual, thus suggesting a limited, if any, role of the\nPDHA1 variant (page 14, lines 385-396).\nauthors provide a detailed interpretation of the position of the mutated amino acids within the\nprotein in the paragraph, for some variants (Glu404Lys, Val673Ala, and Val2338Ile) they do not\nindicate the expected effect of the substitution. For Glu404Lys and Val2338Ile this prediction is\nparticularly important, as they have been classified as benign by in silico predictors.\nAuthor’s reply: After the initial submission of the manuscript, a model of human CELSR1 cadherin\ndomain 1-4 PDB: 8D40 has been submitted to PDB. This structure replaces PDB entries 6C13\nconcerning the Glu404Lys variant.\nFor variants Glu404Lys, Val673Ala and Val2338Ile an extended characterization has been\nincluded in the text. (page 14-17, lines 398-487).\nFigure 2: Arrows are not clearly visible in and their color should be changed to white or black.\nAuthor’s reply: We uploaded an improved and changed figure with black and white arrows, as\nsuggested (Figure 2).\nImage 4: The quality of Image 4 is poor. The name of amino acids and some drawings are\nunreadable, at least in the version of the manuscript submitted for revision.\nAuthor’s reply: We uploaded a high-resolution figure, as suggested (Figure 4).\nFigure 5: The authors do not provide explanations of the meaning of what is shown in panel C.\nAuthor’s reply: Figure 5c depicts the hippocampal commissure phenotype. Coronal sections from\ncontrol (left) and Celsr1 KO (right) newborn mice were immunostained with Tag1 (green) to show\npioneer callosal axons, and GFAP (magenta) to reveal glial cells involved in the midline crossing\nof these axons. Tag1 immunostaining, showed that callosal axons fail to cross the midline and run\nalong the rostral-caudal axis, forming the Probst bundles (pb, Fig. 5c). Glial cells of the indusium\ngriseum (GI, magenta) were absent in the KO. The glial wedge that expresses GFAP and behaves\nas repulsive cue towards extending axons, although present, showed a wider interhemispheric\nspacing (Fig. 5c). This is mentioned in the section results, pages 18, lines 510-522\nFigure 6C: Scale bar should be moved in another place, as it is located on the periventricular zone\nof the brain and does not allow a proper comparison between WT and mutant.\nAuthor’s reply: Thank you. The scale bars were moved in another place (Figure 6).\nCelsr1 KO SVZ compared to controls\". However, they do not provide cells quantification and their\nassumption is probably based on the higher amount of fluorescence observed in mutant brains with\nrespect to WT. The authors should provide cells quantification or change the sentence accordingly.\nAuthor’s reply: We have now provided a quantitative analysis of the BrdU-positive cells and\nrevised Figure 7. The results are shown in the revised Fig. 7h and k. The numbers and p values are\nprovided in the figure legend (Figure 7).\nMinor points\nLine 227: for the sake of clarity, “two brothers of related parents” should be changed to “two sons\nof related parents”.\nAuthor’s reply: Thank you, we changed accordingly (page 11, line 307).\nLine 247 “The EEG showed…”: The sentence should be reworded.\nAuthor’s reply: Thank you, we rephrased the sentence as suggested (page 12, line 328).\nLine 293 “2.2 Molecular characterization of rare CELSR1 biallelic variants”: the title should be\nchanged to “in silico 3D characterization of rare CELSR1 biallelic variants” or somewhat similar,\nas the authors did not perform a real molecular characterization of the variants.\nAuthor’s reply: Thank you, we changed the title as suggested (page 14, line 398).\nline 305 “Based on the crystal structure…”: The sentence should be reworded.\nAuthor’s reply: Thank you, we rephrased the sentence as suggested (page 15, line 413).\nLine 412: \"…been associated brain malformations…\" should be changed to \"…been associated\nwith brain malformations… \".\nAuthor’s reply: Thank you, we changed accordingly (page 20, line 549).\nremoved.\nAuthor’s reply: Thank you, we changed accordingly (page 44, line 1050).\nIn the whole manuscript, panels are indicated in uppercase in the figures and in lowercase in the\ntext and legends.\nAuthor’s reply: Thank you, we changed text, legends and figures consistently.\nSupplementary material, Line 17: “mental retardation” should be changed to “intellectual\ndisability”.\nAuthor’s reply: Thank you, we changed accordingly (supplementary material, page S2).\nAuthors report the clinical features of 8 patients from 6 families with biallelic CELSR1 missense\nvariants. They also performed protein modeling and KO mice studies of the variants. They\nidentified similar findings between human patients and model mice, such as microcephaly, dilated\nlateral ventricles abnormal corpus callosum, and periventricular heterotopia. While CELSR1\nvariants have been reported in patients with lymphedema, neural tube defects, epilepsy, and cardiac\nanomalies, their findings, brain malformations in particular, would expand phenotypes of the\nCELSR1 variants and suggest genotype-phenotype correlation. However, the manuscript needs to\nbe restructured before publication.\nThe descriptions of the Introduction and Discussion are lengthy and too scholastic. They should\nfocus on the original findings of this study.\nAuthors reply: We thank the reviewer for this very useful suggestion. We have rewritten the first\npart of the Introduction, specifying that in our paper we describe a novel neurodevelopmental\ndisorder, with heterogenous phenotypic expression (that include a developmental epileptic\nencephalopathy) associated with CELSR1 variants. Analogously, we have modified significantly\nthe Discussion, highlighting our findings, including novel references, and discussing our results in\nthe light of the data in the literature (page 8-9, lines 163-189)\nThey insist that the brain MRI of Patients #1 and #2 showed “severe” and “diffuse” pachygyria,\nhowever, their occipital lobes appear to be spared in Fig.2A-F. They need to define the meaning of\n“severe” and mark the pachygyria in the occipital region.\nAuthor’s reply: We changed the text accordingly, we removed the term severe and added the\nsparing of the occipital lobes to the legend (page 12, line 318, Fig 2 a-f and supplementary\nmaterial in the patient’s description, pages S2-S3).\nThey mention “diffuse” subcortical white matter abnormalities, however, Fig.2A and D do not\nshow diffuse changes. They look spotty. They also consider the white matter abnormalities as\n“dysmyelination” described in Supplementary material, however they might be gliosis. How did\nthey discriminate gliotic changes from the abnormal intensities in the white matter?\nAuthor’s reply: We changed the text accordingly; we removed the term diffuse and specified the\npossible gliosis. (Page 12, line 318, Table 1 and Supplementary material in the patient’s\ndescription, pages S2-S3).\nbe helpful to show them.\nAuthor’s reply We thank the reviewer for the suggestion. We modified the MRI figure related to\npatient 4 with other images at higher solution. Periventricular nodules are now much more visible.\n(Fig. 2j-l). We changed the legend accordingly. Moreover, some of the previous figures have been\nincluded in the supplementary material, in order to show additional abnormal findings\n(Supplementary material, page S6)\nAs for the patients’ faces, both patients appear to show thin upper lip (vermilion) and bulbous nose\nin Fig.3. Please recheck them.\nAuthor’s reply: we changed the text accordingly (page 13, line 337 and legend of figure 3)\nPatients #1 and #2 showed photosensitivity on EEG. It would be helpful to mention the range of\nphotic stimulation frequency when photoparoxysmal response occurred. Patient #1 showed a\nparoxysmal discharge triggered by eye-closure. It seems to be fixation-off sensitivity. If so, it\nwould be better to briefly mention it.\nAuthor’s reply: Thank you for pointing this out. We have included this sentence in the text:\n“IPS elicited a photoparoxysmal response at frequencies ranging from 6 to 25 Hz (patient #1) and\nfrom 6 to 30 Hz (patient #2)” (page 12, lines 307-308).\nThis information is also included in the descriptions of patients #1 and #2, in the Supplementary\nMaterials (pages S2-S3).\nRegarding the possibility that patient #1 presented a fixation-off sensitivity, this was not\nspecifically investigated (for example with Frenzel lenses). In our laboratory, we perform a 10-sec\nIPS initially with eyes open then, after 5 seconds, we ask the patient to close the eyes and IPS\ncontinues for other 5 seconds with eyes closed. Eye closure is a known way to enhance the effect\nof IPS, and actually in our patient we did not observe a photoparoxysmal response evoked simply\nby eye-closure, without concomitant IPS. Thus, we concluded that we did not have evidence of eye-\nclosure sensitivity.\nPatient #8 appears to show dystrophic changes in muscle biopsy. It would be helpful to show some\nfigures of muscle pathology and discuss the relationships with the CELSR1 variants\nAuthor’s reply: we added a supplementary figure (named Supplementary figure 3), added it to the\nmain text (page 13 lines 346-348). To our knowledge, no relationship between dystrophic changes\nand CELSR1 variants has been demonstrated.\nAuthor’s reply: thank you, we changed the figure and uploaded a more detailed and high-\nresolution image (Figure 4).\nIt would be better to clarify and discuss if and why the radial migration was normal and the\ntangential migration was abnormal in Celsr1 KO mice.\nAuthor’s reply: Radial migration is usually assessed by delayed migration of neuroblasts during\ncortical development, abnormalities in lamination or the in inside out gradient of migration,\nsubcortical band heterotopia, periventricular heterotopia, or extensive migration of neurons and\ninvasion of the marginal zone/layer1. Apart from the periventricular heterotopia, we have not\nobserved any other feature of defective radial migration (Fig. 6). By contrast, the tangential\nmigration of neuroblasts from the subventricular zone to the olfactory bulb was clearly\ncompromised (Fig. 7). This is consistent with previous reports that described a disrupted tangential\nmigration of facial branchiomotor neurons along the rostrocaudal axis of rhombencephalon (Qu\net al., 2010; Glasco et al., 2016; Hummel et al, 2022). While radial and tangential migration share\nseveral features including changes in polarity and cytoskeletal dynamics, they use different\nsubstrates (e.g., glial fibers for radial migration versus chains mode and glial tubes for tangential\nmigration) and are guided by different molecular mechanisms (e.g., Reelin, Lis1, Dcx, Filamin\nversus integrins, semaphorins, neuropilins, cell adhesion molecules, neuregulins, chemokines, slit\nand robo families of attractant and repellent molecules, PSA-N-CAM).\nWhen did Celsr1 KO mice start to exhibit spontaneous seizures?\nAuthor’s reply: Celsr1 KO mice exhibit spontaneous seizures starting from the third postnatal\nweek. This has been specified in the text (page 19, line 530-531).\nPhenotype differences between the previous reports of heterozygous variants and this study of\nbiallelic variants were shown, however, they insist no phenotype-genotype correlations have been\nidentified in the conclusion. They should reconsider it.\nAuthor’s reply: We thank the reviewer for this comment, and we have modified the text accordingly.\nIndeed, literature data show that heterozygous protein truncating or splice site variants leading to\nhaploinsufficiency will likely be associated with lymphedema, at variance with heterozygous or\nhomozygous biallelic missense variants, as in our cohort, which exhibits a primarily\nneurodevelopmental phenotype.\nIn the text, at the end of the Discussion we have included the following sentence:\n8\n“Regarding phenotype-genotype associations our findings, together with the data in the literature,\nsuggest that heterozygous protein truncating or splice site variants leading to haploinsufficiency\nwill likely be associated with lymphedema, at variance with heterozygous or homozygous biallelic\nmissense variants, as in our cohort, which exhibits a primarily neurodevelopmental phenotype.”\n(page 25, lines 677-681). “\nMinor points\nIn the figure legends, IPS and ESES would not need to abbreviate themselves because they appear\nonce in the legends\nAuthor’s reply: we changed the text as suggested (figure 1).\nFig.2H is upside-down.\nAuthor’s reply: thank you, we changed figure as suggested (figure 2).\nCase reports in the supplementary material should be described in the past tense.\nAuthor’s reply: we changed the text as suggested (supplementary material).\nThe term, “mental retardation”, should be changed to “intellectual disability”.\nAuthor’s reply: we changed the text as suggested (supplementary material).\nDelete the year when WES was performed in each patient. It is unnecessary to enhance medical\naccuracy for this study.\nAuthor’s reply: we deleted this information (supplementary material).\nESES usually means “Electrical” status epilepticus in sleep [Specchio N, et al. Epilepsia\n2022;63:1398-1442]. Not “Encephalopathy related to”.\nAuthor’s reply: We thank the reviewer for raising this point. Indeed, the term ESES has been used\nto label either the clinical picture (i.e. the encephalopathy related to status epilepticus during\nsleep) or the EEG picture (i.e. the electrical status epilepticus during sleep) (these aspects have\nbeen discussed in Rubboli et al.. Epilepsy and Behavior, Epilepsy Behav. 2023;140:109105). We\nacknowledge that recently the term of DEE-SWAS has been introduced to encompass all those\n9\nconditions with a striking enhancement of epileptic EEG activity during sleep associated with a\nbehavioral-cognitive derangement, including ESES (Specchio et al., Epilepsia. 2022\nJun;63(6):1398-1442. doi: 10.1111/epi.17241. Epub 2022 May 3.2022). However, in the paper we\nhave chosen to use both terms, i.e. ESES/DEE-SWAS, since we think that ESES has still a specific\nnosologic place within DEE-SWAS (Rubboli et al., Epilepsy Behav. 2023 140;109105), and also,\nbecause ESES is still widely used among the professionals. We hope that this can be acceptable by\nthe editors and the reviewers.\nCPK should be CK in formal and would be better spelled out.\nAuthor’s reply: we changed the text as suggested (supplementary material).\nIn Supplementary Table 1, they classify the variants as III or IV, possibly based on the ACMG\nguideline. The original ACMG guideline published in 2015 mentions i) pathogenic to v) benign.\nDo they mean IV is likely benign?\nAuthor’s reply: Thank you for the reviewer remark. According to ACMG guidelines classes are\ndescribed from I to V, ranging from benign to pathogenic respectively. In our paper only class III-\nV variants are reported for which class III variants are defined as “variant of unknown clinical\nsignificance”, class IV as “likely pathogenic” and class V as “pathogenic” variants. We added\nthis in the text (page 26, lines 715-717).\nThe authors report the phenotype of 8 individuals from 6 unrelated families with bi-allelic variants\nin CELSR1. Pathogenicity and functional effects of the variants were inferred by bioinformatic\nanalysis, protein modelling and prediction tools. Further support for the role of CELSR1 in\nneurodevelopment was provided by description of the clinical phenotype and analysis of the adult\nbrain of Celsr1 KO mice.\nThis report significantly adds to the current knowledge on brain development and deserves\npublication.\nThere is one major point I would like the authors to address prior to publication, which is statement\nthat bi-allelic variants in CELSR1 are a cause of Developmental and Epileptic Encephalopathy\n(DEE).\nIn the introduction, the authors include the definition of DEE as follows “Developmental and\nEpileptic Encephalopathies (DEE) form a group of severe childhood onset disorders characterized\nby neurodevelopmental regression or plateauing often associated with early onset refractory\nepileptic seizures. Epileptic activity “per se” can contribute to severe cognitive and behavioral\nimpairment, above what is expected from the underlying pathology, and the developmental\nconsequences may improve with the amelioration of the epileptiform activity.” In the CELSR1\ncohort reported here, only 1 out of 8 individuals (patient #5) showed a neurodevelopmental\nregression in the context of a DEE-SWAS. Only 2 additional individuals had epilepsy which did\nrespond to anti-seizure medication. I therefore disagree to classify the phenotype associated with\nbi-allelic variants as an example of a DEE based on the information available to date. In line with\nthis view, I would suggest to adapt the introduction with less focus on DEE and rephrase the\nstatements linked to DEE throughout the rest of the manuscript, including the abstract.\nAuthor’s reply: We thank the reviewer for this important comment. Indeed (also to comply with the\ncomment of Reviewer 1), we have rewritten the first part of the Introduction specifying that in our\npaper we describe a neurodevelopmental disorder with several features including epilepsy in some\nsubjects. In our cohort, only one patient exhibited a DEE (i.e. ESES/DEE-SWAS) We have modified\nthe whole manuscript accordingly (page 8, lines 163-187).\nI have a few minor suggestions that might improve the manuscript.\nI would propose to include a sentence on the high degree of phenotypic variability in this cohort\nin the abstract and to highlight this a bit more in the discussion. Brain abnormalities were indeed\npresent in most individuals but the type of abnormality varied considerable between individuals.\nThe degree of developmental impairment was also highly variable ranging from one child dying\n11\nin infancy to two adolescents with much milder phenotypes. Epilepsy was present in only 3 out of\n8, again with variable seizure types and response to treatment.\nAuthor’s reply: Thank you for the suggestion we have modified the Abstract accordingly and\nincluded a sentence in the Discussion (abstract and page 20, lines 544-550):\n“These phenotypic features showed a high degree of variability, in particular NDD and ID could\nrange from mild to moderate or very severe, language could vary from normal to severely delayed,\ndysmorphisms were present only in half of the patients as well as epilepsy who affected only 3 out\nof 8 individuals. Finally, various types of brain malformation were observed”.\nAbstract: include white matter changes as part of the brain abnormalities.\nAuthor’s reply: Thank you. Done (page 7, line 144)\nAbstract: If word count allows, I would propose to include the increased susceptibility to seizures\nin the Celsr1 KO mice since this provides an additional link to part of the human phenotype.\nAuthor’s reply: Thank you. Done (page 7, line 151)\nIntroduction: “CELSR1 is key to ependymal cilia polarization and ciliary beats and its loss\nseverely disrupts circulation of the cerebrospinal fluid and results in hydrocephalus. Variants in\nthe CELSR1 gene have been linked to congenital defects in the vestibulocochlear system, heart,\nkidney, and lung.” Based on these findings, could the CELSR1-related phenotype be classified as\na ciliopathy? Arguments in favour/against?\nAuthor’s reply: Different phenotypes such as that of ependymal cilia, vestibulocochlear system and\nurogenital tract were associated to ciliary dysfunctions. Others like neuronal migration,\nabnormalities in the heart, kidney, and lung have not been linked to cilia to the best of our\nknowledge. These phenotypes, shared with other PCP genes, have not been considered as\nciliopathies per se, so we preferred not to qualify them as such.\nIntroduction: “In the developing rhombencephalon, CELSR1 specifies the directionality of\ntangential migration of facial nerve motor neurons. CELSR1 is also key to ependymal cilia\npolarization and ciliary beats and its loss severely disrupts circulation of the cerebrospinal fluid\nand results in hydrocephalus.” It would be helpful if you could mention if these conclusions were\nbased on work in mice or other models.\nparagraph by line break to describe the mouse phenotype (page 10, lines 254-260).\nResults: Epilepsy phenotype – replace ESES by D/EE-SWAS as per latest ILAE report? Idem for\nTable and Case Description in Supplementary Materials.\nAuthor’s reply: We thank the reviewer for raising this comment. Indeed, the term ESES has been\nused to label either the clinical picture (i.e. the encephalopathy related to status epilepticus during\nsleep) or the EEG picture (i.e. the electrical status epilepticus during sleep) (these aspects have\nbeen recently elaborated in Rubboli et al.. Epilepsy and Behavior, Epilepsy Behav.\n2023;140:109105). We acknowledge that recently the term of DEE-SWAS has been introduced to\nencompass all those conditions with a striking enhancement of epileptic EEG activity during sleep\nassociated with a behavioral-cognitive derangement, including ESES (Specchio et al., Epilepsia.\nthe comment of reviewer #2, in the paper we have chosen to use both terms, i.e. ESES/DEE-SWAS,\nsince we think that ESES has still a specific nosologic place within DEE-SWAS (Rubboli et al.,\nEpilepsy Behav. 2023 140;109105), and also, because ESES is still widely used among the\nprofessionals. We hope that this can be acceptable by the editors and the reviewers.\nDiscussion: “Pachygyria is included in the larger group of lissencephalic syndromes, and it is\ncharacterized by decreased cortical convolutions, cortical thickening, and a smooth cerebral\nsurface.” More recent references to the classification of lissencephaly include PMID 28440899\nand PMID 29671837.\nAuthor’s reply: Thank you for the suggestion, we have incorporated the suggested references in\nthe revised text (page 20, line 555).\nDiscussion: “Pathogenic variants in ACTB and ACTG1 genes have also been associated with\npachygyria.” Not relevant to refer to only these two genes. not relevant to highlight only these two\ngenes. Pachygyria a>p can be seen with variants in DCX, ACTB, DYNC1H1, ACTG1, KIF5C,\nVLDLR, CRADD, or ARX (in that case with ACC). Reference PMID: 32895508.\nAuthor’s reply: Thank you for the suggestion, we have incorporated the suggested references in\nthe revised text (page 20, line 555-556).\nDiscussion: “PNH consists of nodules of grey matter located along the lateral ventricles with a\ntotal failure of migration of some neurons.” More accurate reference includes PMID: 32895508.\nthe revised text (page 20, line 556, 561, 562).\nDiscussion: Three individuals have abnormal corpus callosum. It is unclear from the images and\ntable which patients are concerned. Patient 4 has a convincingly short and thick corpus callosum.\nPatient 3 is reported to have a course small corpus callosum in the Table, but this is not clear in\nFigure 3I. There is no third individual mentioned with callosal abnormalities in the Table.\nAuthor’s reply: thank you for pointing this out. There are two patients with corpus callosum\nabnormalities (patient 3 and 4). We modified the table accordingly (page 12, line 319-323, table 1\nand table 2).\nFigure 2\nFigure 2C: Cerebellar vermis hypoplasia?\nAuthor’s reply: thank you for pointing this out. Our neuroradiologist reviewed the entire MRI and\ndoes not confirm the hypoplasia. In particular the size of the pons of this patient is normal, thus\nconfirming the absence of abnormalities of the vermis cerebellaris (figure 2).\nFigure 2G-I: patient #3 – Since I do not have access to the entire MRI it is difficult to judge the\ncortical pattern but based on Fig2H I have the impression that the gyration is not normal either in\nthis individual.\nAuthor’s reply: thank you for pointing this out. Our neuroradiologist reviewed the entire MRI and\nchanged the description and the figures, choosing better resolution imaging and describing in\ndetail the alterations of this patient (#3) that include: focal gray matter heterotopia in the right\nlateral ventricle and non-specific abnormalities, such as a coarse and small corpus callosum, and\nenlarged lateral ventricles. We removed the alteration of the cortical pattern (page 12, lines 319-\n320, figure 2 legend, table 1 and supplementary materials).\nFigure 2J: The periventricular nodular heterotopia are unclear on this image. Would it be an option\nto provide an image in the axial plane to illustrate the PNH?\nAuthor’s reply We thank the reviewer for the suggestion. We modified the MRI figure related to\npatient 4 with other images at higher solution. Periventricular nodules are much more visible.\n(Fig. 2j-l). We changed the legend accordingly. Moreover, some of the previous figures have been\nincluded in the supplementary material, in order to show additional abnormal findings\n(Supplementary material, Page S6)\nAuthor’s reply: thank you for pointing this out. Our neuroradiologist reviewed the entire MRI and\ndoes not confirm alterations of the cortical pattern, whereas the imaging confirms cerebral\natrophy. We added this finding in the text. (text page 12, lines 324, figure 2 legend, table 1 and\nsupplementary materials).\nTable 1: head circumference now either by centile or standard deviation. Since the definition of\nmicro/megalencephaly is based on SD, it would be preferred to have all SD for all head\ncircumference measures.\nAuthor’s reply: thank you, we changed the table as suggested (table 1).\nSupplementary material:\nPatient #1: please replace ‘mental retardation’ by ‘intellectual disability’\nAuthor’s reply: thank you for the suggestion, we have changed the text accordingly (supplementary\nmaterial, page S2).\nPatient #6: autism spectrum disorder, level 2 – unclear what ‘level 2’ stands for.\nAuthor’s reply: we removed the classification for both patient #6 and #7 both in table 1 and in the\nsupplementary material (page S8-S9).\nPatient #8: “Later a trio WES and whole genome sequencing (WGS) were performed and disclosed\ntwo missense variants, c.1210G>A (p.Glu404Lys) and c.4415C>G, (p.Thr1472Ser) in CELSR1\nwith maternal and paternal inheritance, respectively.”\nAuthor’s reply: we modified the text as suggested (page S10).\nThe description of the structural prediction is insufficient and the validity is unclear.\nFirst, it is necessary to properly describe how multiple prediction tools are used, along with the\nreasons. It is also necessary to describe all the options used to predict each tool (for example, the\nprediction results of Phyre2 differ between Normal mode and intensive mode).\nIn homology modeling, the reliability of the prediction varies greatly between the regions covered\nby the template and those outside the template. Therefore, it is necessary to provide information\nabout the template and sequence alignment used for all predictions. E-values are calculated from\nthe entire sequence alignment and do not guarantee local quality, so they are insufficient for the\ncurrent discussion.\nIt is also necessary to discuss the quality of the template structure. For example, PDBID:6C13 is\nan EM structure with a resolution of 11.3 angstroms, and it is considered to be an inappropriate\ntemplate for the current discussion.\nAuthor’s reply: Concerning options used for the structural predictors, this point has now been\naddressed in paragraph 4.3 (page 27, lines 730-739).\nConcerning criteria for sequences alignments this point has now been addressed in the beginning\nof paragraph 4.3. Hence, E-values are solely concerning local alignment.\nConcerning PDBID:6C13. Since the initial submission of the manuscript, a model of human\nCELSR1 cadherin domain 1-4 PDB: 8D40 has been submitted to PDB. This structure replaces\nPDB entries 6C13 concerning the Glu404Lys variant.\nThis study describes the phenotype of eight individuals with biallelic variants of CELSR1,\npresenting a novel epileptic encephalopathy characterized by brain malformations,\nneurodevelopmental delay, behavioral disorders, and epilepsy. Whole exome sequencing revealed\ncompound heterozygous and homozygous variants. Analysis of Celsr1 mutant mice showed\nphenotypes similar to those found in humans, such as partial agenesis of the corpus callosum,\nperiventricular heterotopia, and enlarged lateral ventricles, highlighting the critical role of CELSR1\nin human neurodevelopment. While not finding an exact parallelism between mouse and human\nphenotypes, mutant mice for this gene could serve as a model for studying this disorder.\nInterestingly, the authors suggest that potential treatment with retinoic acid administered during\npregnancy could help alleviate this condition in the future. It is an intriguing study but there are\nseveral issues that need to be addressed before publication:\n1. Since most patients are heterozygous, it would be interesting to know to what extent\nheterozygous mice exhibit similar phenotypes.\nAuthor’s reply: Heterozygous mice have no noticeable phenotype, and all our patients don’t have\nheterozygous but rather biallelic mutations in the CELSR1 gene.\nIn the literature, it is not unusual that heterozygous mutations in mice do not faithfully recapitulate\nthe human mutations. In most cases, humans are more sensitive than mice to heterozygous\nmutations. Examples include the mice bearing a heterozygous mutation in the DCC gene which\nhave almost no phenotype (Thiry et al, 2022\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8906791/), which contrasts with the strong\nphenotype described in patients with heterozygous (Schott and Wyke, 1981; Srour et al., 2010), or\nbiallelic mutations (Jamuar et al., 2017). Discrepancies between patients and mouse models have\nbeen reported since 1990s-2000s (see for instance Elsea and Lucas 2002. The mousetrap: what\nwe can learn when the mouse model does not mimic the human disease,\nhttps://pubmed.ncbi.nlm.nih.gov/11917158/).\n2. Line 361: Although the authors state that they focus on the analysis of adult brain, many analyses\nare actually conducted on newborn animals.\nAuthor’s reply: Thank you for pointing that out. We have modified the text and changed “adult\nbrain” into “postnatal brain” (page 18, line 487).\n3. Why do they analyze P0 mice in Figure 5 and P18 mice in Figure 6? It would be desirable to\nanalyze both ages in both cases.\naccording to the finding we intended to illustrate, we used the most adapted stage and approach.\nFor instance, we used the mature brain (Fig. 5a and b) to show the end result of the defective\ncorpus callosum, and P0 brain (Fig. 5c and d) to trace callosal pioneer axons as they navigate\ntowards their destination and investigate the cellular/molecular mechanisms involved in the\nmidline crossing (Figure 5 and 6).\n4. In panels with high-magnification pictures, it is challenging to identify the specific brain area.\nIncluding drawings or lower-magnification photographs indicating the specific area shown in the\nhigh-magnification photo would be helpful.\nAuthor’s reply: Thank you for flagging this. We have included low magnification photographs and\nindicated the analyzed areas. (Figure 5).\n5. Figure 6: What is the conclusion of the BrdU injection experiments? Is there increased\nproliferation in mutants? These results should be quantified. Additionally, the control photo does\nnot clearly show BrdU-positive cells because the ventricle edge is not visible.\nAuthor’s reply: The conclusion of the BrdU injection experiments is that a proportion of the Celsr1\nmutant neuroblasts fail to leave the ventricular zone and migrate to the cortical plate. This is most\nlikely due to an increased proliferation as described in Boucherie et al., 2018. This has been\nquantified and shown in revised Fig. 6g. The control figure has been shifted to make the ventricle\nedge visible (figure 6).\n6. Figure 7: i/The expression of Celsr1 in the olfactory bulb is intriguing, but as it is a null mouse\nline, correlating this expression with observed defects may not be valid. Defects could be due to\ndevelopmental issues.\nAuthor’s reply: We agree that olfactory bulb phenotype could be due to defects during embryonic\ndevelopment as we use constitutive KO mice. We believe however that this is unlikely for 2 reasons:\nFirst, the tangential migration of neuroblasts to olfactory bulb occurs mainly in the postnatal\nbrain. Second, we didn’t observe abnormality at birth and the first signs of a reduction of the\nolfactory bulb appear only after 1 month. We added this discussion in the text: “It may be argued\nthat olfactory bulb phenotype could be due to defects during embryonic development as we use\nconstitutive KO mice. We believe however that this is unlikely for two reasons: (i) the tangential\nmigration of neuroblasts to olfactory bulb occurs mainly in the postnatal brain; (ii) we did not\nobserve abnormality at birth and the first signs of a reduction of the olfactory bulb appear only\nafter one month after birth”. (page 23, line 638-643).\n18\nii/Including drawings or lower-magnification sections indicating the photographed area in panels\nD and E would be helpful. iii/ Adding labels with the names of the structures shown would also\naid comprehension.\nAuthor’s reply: Thank you for pointing this out. We have included low magnifications photographs\nof brain sections stained with Cresyl violet and delineated the analyzed areas. We have also added\nthe names of key brain structures to help comprehension (figure 7).\n7. Line 396: The word \"show\" in \"Dcx immunostaining show that neuroblast migration is\ndisrupted...\" should be replaced with \"suggest.\"\nAuthor’s reply: Done (page 19, line 525).\n8. The epilepsy results in the Supplementary Table should not be supplementary data but included\nin the main text, as they represent a phenotype mimicking that of humans\nAuthor’s reply: Done. Thank you (page 19, lines 530-531).\nPoint-by point reply to reviewers\nREVIEWER COMMENTS\nReviewer #4\n1. I asked the authors how multiple prediction tools are used. It includes a question of how to\nselect a model from multiple prediction models. However, the authors only wrote \"When two or\nmore modelers predicted a model this was selected.\" and it is impossible to understand the\nselection criteria etc.\nReply: We apologies for not having provided the information requested by the reviewer. In this\nfurther revised version, the following text has been added to section 4.3, line 642-645: “When two\nor more modelers predicted a model this was selected based on highest template-model score if\nadditionally supported by sequence similarity based on multiple sequence alignment (MSA) with\nidentity above 30% and functional homology of the template. Templates with a resolution below\n4.5 Å and a R value below 0.25 were selected.”\n2. In the author's response, they wrote that \"Concerning criteria for sequences alignments\nthis point has now been addressed in the beginning of paragraph 4.3. Hence, E-values are solely\nconcerning local alignment.\" However, I cannot find any description of the point in paragraph 4.3.\nReply: E-values and probabilities were included in the previous version of the manuscript. In this\nrevised version, in section 4.3 line 643, we report that the alignment should be more than 30% (as\nspecified in point 1 above). We have added the following sentence (lines 643-645): “…if\nadditionally supported by sequence similarity based on multiple sequence alignment (MSA) with\nidentity above 30% and functional homology of the template”.\nPDB states “Proteins with similar amino acid sequences (more than ~30% sequence identity) are\nknown to fold into similar structures“.\nIn addition the following text has been added to section 4.3, lines 636-641: “The SMART server\n(http://smart.embl-heidelberg.de/) was used for assessment of the functional domains of CELSR1.\nThe structure of each functional domain was obtained individually performing analysis for\nstructural homology using HHPRED67 (https://toolkit.tuebingen.mpg.de), Phyre268\n(http://www.sbg.bio.ic.ac.uk/phyre2), I-TASSER69”\n1\n3. I require a discussion about the quality of the template structures used in this research. Without\na careful check, inappropriate selections like 6C13 will occur. If there are some conditions in the\nselection, the authors should denote them properly.\nReply: Thanks for raising this issue, however we wish to specifiy that we have not used 6C13.\nThe following text has been added to the discussion (lines 526-529): “The predicted models are\nconsidered highly reliable based on quality of the templates used, the estimate of the modeling\nmatch, functional similarities and the overall sequence similarity between the template and the\nCELSR1 domain under investigation”.\nIn addition in the Section 2.2 “In Silico characterization of rare CELSR1 biallelic variants”,\nline\n395, we now refer to a new table, Supplementary table 2, specifying the quality parameters of the\ntemplates used. These are all “excellent” to “good”.\nReviewer #5\nThe authors have satisfactorily answered my questions and concerns. The only point where they\ncould improve is the first one. I think they should clearly state in the text the fact that heterozygous\nmice have no noticeable phenotype, and most of the patients don’t have heterozygous but rather\nbiallelic mutations in the CELSR1 gene.\nReply: we thank the reviewer for this comment. We have added this sentence in the Results section,\nlines 405-406 “Heterozygous mice were indistinguishable from controls. Celsr1 KO mice exhibited\nagenesis of the caudal corpus callosum” and this sentence in the Discussion, lines 557-559:\n“Heterozygous mice have no noticeable phenotype. The KO mice have a complete loss of CELSR1\nfunction and exhibit the full phenotype.”\n2"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have dealt with all the points I have raised.", "reviewer_2": "The authors have provided comprehensive and appropriate responses to the reviews of the previous version of the\nmanuscript. A minor suggestion is raised as follows,\n“Developmental epileptic encephalopathy” should be changed to “Developmental and epileptic encephalopathy”.", "reviewer_3": "I have reviewed the revised version of this manuscript and am happy with the current version.", "reviewer_4": "The authors revised a paragraph about protein structural modeling. However, they have not responded to critical points and\nthe revision is inadequate.\n1. I asked the authors how multiple prediction tools are used. It includes a question of how to select a model from multiple\nprediction models. However, the authors only wrote \"When two or more modelers predicted a model this was selected.\" and\nit is impossible to understand the selection criteria etc.\n2. In the author's response, they wrote that \"Concerning criteria for sequences alignments this point has now been\naddressed in the beginning of paragraph 4.3. Hence, E-values are solely concerning local alignment.\" However, I can not\nfind any description of the point in paragraph 4.3.\n3. I require a discussion about the quality of the template structures used in this research. Without a careful check,\ninappropriate selections like 6C13 will occur. If there are some conditions in the selection, the authors should denote them\nproperly.", "reviewer_5": "The authors have satisfactorily answered my questions and concerns. The only point where they could improve is the first\none. I think they should clearly state in the text the fact that heterozygous mice have no noticeable phenotype, and most of\nthe patients don’t have heterozygous but rather biallelic mutations in the CELSR1 gene."}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_4": "The revised manuscript is now acceptable."}, "author_response": ""}]

Decision letter