CMIP as a novel candidate gene for neurodevelopmental and neuropsychiatric disorders

CMIP as a novel candidate gene for neurodevelopmental and neuropsychiatric disorders | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CMIP as a novel candidate gene for neurodevelopmental and neuropsychiatric disorders Frank Kooy, Matthias De Wachter, Mathijs van der Lei, Amber Decleve, and 30 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8316803/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract CMIP , a c-maf inducing protein that plays a key role in cytoskeletal remodeling, neuronal migration and synaptic formation, was first associated with specific language impairment and autism through the identification of a deletion in a single patient in 2012. Since then, only two additional individuals with CMIP deletions have been reported, both sharing features of autism and gastrointestinal features. However, a firm causal relationship between variants in CMIP and neurodevelopmental disorders has not yet been established. In this multicentre cohort study, we identified 25 individuals with CMIP -related neurodevelopmental disorders, 22 of whom have not been previously reported. Of these, seven individuals carried heterozygous loss-of-function CMIP single nucleotide variants, while the other 18 individuals had a complete or partial deletion of CMIP , some involving adjacent genes. The clinical phenotype was variable with a high prevalence of developmental delay (20/25), autism spectrum disorder features (13/25), attention-deficit/hyperactivity disorder features (11/25) and other psychiatric disorders (15/25). Epilepsy was present in nine individuals (9/25), of whom three had therapy-resistant seizures. To study the pathogenicity of CMIP variants, a cmip mutant zebrafish model carrying a premature stop codon was investigated. These mutants showed temperature-dependent altered locomotor activity suggestive of seizure-like behavior, which was confirmed by spontaneous epileptiform discharges in cmip +/− mutant zebrafish larvae. Our patient cohort and the zebrafish data establish CMIP as a gene implicated in neurodevelopmental and neuropsychiatric disorders. We recommend inclusion of CMIP in the genetic work-up of neurodevelopmental delay, with or without autism or psychiatric disorders and epilepsy. Health sciences/Diseases/Neurological disorders/Neurodevelopmental disorders/Autism spectrum disorders Health sciences/Diseases/Psychiatric disorders Health sciences/Diseases/Neurological disorders/Epilepsy autism spectrum disorder psychiatric disorder language delay intellectual disability epilepsy zebrafish Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction CMIP ( c-maf inducing protein ), located on chromosome 16q23.2, encodes a multi-domain adaptor protein composed of an N-terminal pleckstrin homology domain, a central region with multiple protein interaction motifs, and a C-terminal leucine-rich repeat domain. 1 It is expressed mainly in the brain and in other tissues, including increased expression in peripheral mononuclear blood cells and kidney. 2 In the central nervous system, CMIP modulates network excitability mainly by repressing cortical interneuron activity, a mechanism increasingly recognized in the pathophysiology of neurodevelopmental and neuropsychiatric disorders. 3 – 5 Protein interactors of CMIP include the RelA subunit of nuclear factor kappa-B (NF-κB) transcription factor , 6 the p85 regulatory subunit of phosphoinositide-3 kinase (PI3K), and the death activated protein kinase-interacting protein 1 (Dip1), 1 linking CMIP to multiple signaling pathways relevant to neural development and immune function. It also interacts with filamin A, a key player in neuronal migration. 7 , 8 The PI3K/Akt/mTOR pathway is a central regulator of neural progenitor proliferation, neuronal differentiation, migration, axon growth and synaptic plasticity. Disruption of this pathway during critical periods of brain development can lead to abnormal cortical architecture, imbalanced excitatory/inhibitory (E/I) signaling and impaired synaptic maturation features commonly observed in neurodevelopmental disorders (NDD) and autism spectrum disorder (ASD). 9 – 11 Genome-wide association studies (GWAS) have identified CMIP as a candidate gene for specific language impairment (SLI), reading abilities and general cognitive function. 12 – 15 In this context, CMIP emerged as a potential candidate gene for NDD following two independent case reports, in which ASD and developmental delay (DD) were the most striking features. These individuals had a complete or partial deletion of CMIP , in combination with adjacent genes at chromosome 16q23. 16 , 17 The first reported case presented with global DD during infancy and febrile seizures. Over time language acquisition was more significantly impaired and the child was diagnosed with ASD. 17 In the other publication, including two independent cases, a similar clinical phenotype was observed, accompanied by gastrointestinal symptoms, suggesting a syndromic form of ASD. 16 Despite these findings, the link between CMIP and NDD and/or neuropsychiatric disorders remained a matter of debate. In this study, we outline the pathogenicity of CMIP through a multicentre case series of 25 individuals with CMIP -related NDD, alongside functional assessment in a CMIP loss-of-function zebrafish mutant model. Together, these data define the phenotypic spectrum associated with CMIP variants and support its classification as a novel NDD gene. Materials (Subjects) and methods Study design and collection of clinical characteristics This multicentre, retrospective observational study described clinical features of individuals with CMIP -related neurodevelopmental disorder, recruited through GeneMatcher and DECIPHER between 2020 and 2025. 18 , 19 Data were collected using a standardized datasheet provided to participating clinicians (supplemental material). Genetic analyses were performed according to local protocols. Epilepsy was defined and classified according to the 2022 classification of the International League Against Epilepsy (ILAE). 20 , 21 Developmental delay was defined as failure to meet expected developmental milestones in one or more domains. Intellectual disability (ID) was defined according to DSM-4 and DSM-5, based on formal intelligence coefficient testing,if available (normal: ≥ 85, borderline: 70–84; mild 55–69; moderate 40–54; severe or profound < 39). 22,23 When testing was unavailable, classification relied on clinical records and required support level.The manuscript followed STROBE guidelines supplemental material). 24 Review of the literature To outline the phenotypical spectrum of individuals with a CMIP -related NDD, a PubMed literature search (September 2025) was performed using the terms: (CMIP) AND ("neurodevelopmental disorders"[MeSH Terms] OR (neurodevelop* disorder*[Tiab]) OR ("child mental disorder*"[Tiab]) OR ("intellectual disab*"[Tiab]) OR ("developmental delay"[Tiab])), identifying eight articles and two additional individuals [22]. Zebrafish model and husbandry Heterozygous cmip zebrafish (sa43067), carrying a premature stop codon (g.13351012A > T), were obtained from the European Zebrafish Resource Center and originally generated by ENU mutagenesis at the Stemple Lab. Adult fish were maintained at 28.5°C in UV-sterilized water on a 14/10h light/dark cycle under standard aquaculture conditions. Fertilized embryos were collected via natural spawning and raised at 28°C in embryo medium (1.5 mM HEPES, pH 7.2, 17 mM NaCl, 2 mM KCl, 0.12 mM MgSO4, 1.8 mM Ca(NO3)2, 0.6 µM methylene blue). Behavioral analysis Cmip zebrafish larvae (7 dpf) were transferred individually into 96-well plates containing 100 µl Danieau’s medium, and behavior was monitored for 60 minutes in darkness at 28°C or 32°C using DanioVision™ (Noldus, The Netherlands). The behavioral metrics are calculated for every 5-min period. Ethovision XT16 (Noldus, Netherlands) was used to quantify locomotor behavior as total distance moved (mm) per 5 min and as cumulative duration in highly active state (s) per 5 min Activity was defined as pixel changes between consecutive images, ranging from 0% (no change) to 100% (all pixels changed). The highly active state has previously been associated with seizure-like behavior. 25 Non-invasive local field potential (LFP) recordings Non-invasive LFP recordings of 7 dpf larvae were performed as previously described. 26 – 29 Signals were recorded from the optic tectum for 10 minutes at room temperature using glass electrodes filled with artificial cerebrospinal fluid (124 mM NaCl, 2 mM KCl, 2 mM MgSO4, 2 mM CaCl2, 1.25 mM KH2PO4, 26 mM NaHCO3 and 10 mM glucose). Epileptiform events were defined as discharges ≥ threefold baseline amplitude and ≥ 50 ms duration and quantified using Clampfit 10.2 (Molecular Devices Corporation, USA). Genotyping Genomic DNA was extracted from 7 dpf larvae andlysed with 50 mM NaOH at 95°C for 10 minutes and neutralized with Tris-HCl (pH 8.0). Genotyping PCR was performed using GoTaq® G2 DNA Polymerase (Promega) with custom primers (Table S1 ) at 58°C. Amplicons were analyzed by QIAgel capillary electrophoresis and Sanger sequencing and aligned to the cmip reference genome using CLC Genomics Workbench 5. RNA extraction and RT-PCR RNA extraction and RT-PCR RNA was extracted from pooled larval brains (n = 5–10 per genotype) using the RNeasy Micro Kit (Qiagen). RNA concentration and purity were measured using Qubit™ RNA assay (Invitrogen™) and NanoDrop™ Spectrophotometer (Thermo Scientific). For RT-PCR, 1 µg of RNA was reverse-transcribed using the Superscript III First-Strand Synthesis System (Invitrogen) and primer efficiencies were optimized using standard dilution curves. RT-PCR was performed in triplicate with SYBR Green I Mastermix (Eurogentec) on a CFX384 Touch system (BioRad, USA), with primers listed in Table S1 . Data were analyzed in in QBASE+ (Biogazelle, Belgium), using Ube2a, Rpl3 and Hatn10 as reference genes. Western blotting Protein was isolated from pooled zebrafish larvae brains (n = 5–10 per genotype) and homogenized using a TissueRuptor II (Qiagen) in ice cold RIPA buffer (150 mM NaCl, 50 mM Tris, 0.5% sodium deoxycholate, 1% NP-40 and 2% SDS) supplemented with protease (cOmplete™ Mini, Roche) and phosphatase (PhosSTOP™, Roche) inhibitors. Lysates were incubated on ice for 15 minutes and cleared by centrifugation for 10 minutes at maximal speed. Protein concentration was determined with the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (7µg) were mixed with NuPAGE™ Sample Reducing Agent (Invitrogen) in Laemmli buffer (Bio-Rad), heated at 70°C for 10 min, and separated on Bolt™ 4–12% Bis-Tris gels (Invitrogen) in MOPS buffer. Proteins were transferred onto nitrocellulose membranes (Cytiva) using a Mini Trans-Blot® Cell (Bio-Rad). Transfer efficiency was verified by Ponceau S staining (Sigma). Membranes were blocked in 5% non-fat dry milk (Carl Roth) in TBST for 1 hour at room temperature, followed by overnight incubation at 4°C with primary CMIP antibody (Proteintech 12851-1-AP, UK; 1/1000. HRP-conjugated immunoglobulins (Agilent Santa Clara,, USA; 1/2000) were appliedunder identical conditions. Signals were detected using SuperSignal™ West Pico PLUS (Thermo Scientific),. with West Femto used for CMIP to enhance sensitivity Images were acquired with an Amersham™ Imager800 (Cytiva, USA), using GAPDH (Cell signaling technology, USA) as loading control and quantifiedusing ImageJ/Fiji software. Statistical analysis Clinical data were analysed using IBM SPSS Statistics software, version 29 (IBM Corp., Armonk, NY, USA). Normally distributed values were reported as mean ± SD, non-normally distributed values were represented as median (Q1-Q3). median with quartile 1 (Q1) and 3 (Q3). Zebrafish dataare presented as mean ± SEM. Statistical analyses were performed using one- or two-way ANOVA with with Dunnett’s post hoc test or Kruskal–Wallis test with Dunn’s multiple comparisons. Outliers were identified via the ROUT test (Q = 0.5%) and excludedin GraphPad Prism 10 (GraphPad Software, USA). Results Clinical and molecular characteristics We identified 22 previously unreported individuals with CMIP -related neurodevelopmental disorders and updated clinical and genetic data for three previously published cases, including one reported by our group (Table 1). Median age at last evaluation was 15 years (range: 1.3-64 years) 14 individuals were male and 11 were female. Altogether, these 25 individuals originated from 18 unrelated families. Six different truncating single nucleotide variants (SNVs) were present in seven of the 25 individuals (28%). The remaining 18 individuals (72%) had a complete or partial deletion of CMIP , which also included additional protein coding genes (Fig. 2). These deletions ranged between 283 kb and 6,97 Mb. In 12/18 cases (67%), the entire CMIP gene was deleted. Additional protein coding genes included in the deletions are: ADAD2, ATMIN, ATP2C2, BCO1, C16orf46, CDH13, CDYL2, CENPN, CIBAR2, CLEC3A, CMC2, COTL1, CRISPLD2, DNAAF1, DYNLRB2, GAN, GCSH, GSE1, HSBP1, HSD17B2, HSDL1, KCNG4, KIAA0513, KLHL36, MAF, MBTPBS1, MEAK7, MLYCD, MPHOSPH6, NECAB2, OSGIN1, PKD1L2, PLCG2, SDR42E1, SCL38A8, TAF1C, USP10, WFDC1, WWOX and ZDHHC7 , based on genomic coordinates (hg19: chr16:77,983,154 - 85,673,761), spanned from the most proximal to the most distal. A detailed overview of the CMIP deletions and the affected protein coding genes is provided in Table S2. Perinatal history Ten out of 25 individuals (40%) were born premature, between 26 and 37 weeks of gestational age. Birth biometry was unremarkable, apart from case s 20 and 21 who were large for gestational age, and case 24 who was small for gestational age. Neonatal manifestations included respiratory or feeding features , but all individuals were discharged without major complications. Case 10 underwent surgical intervention to repair a complex congenital heart defect. In 4/25 (16%) individuals hypotonia in the perinatal period was reported. Clinical examination Dysmorphic facial features were reported in 8/25 (33%) of individuals. Macrocephaly was observed in three individuals . Frontal bossing, a broad forehead, and a prominent nose were reported in multiple families. However, no distinctive dysmorphic features could be identified. Variable abnormalities including balance abnormalities and movement disorders were seen at neurological examination in 12 out of 25 individuals (48%). Two individuals from the same family (family 4) developed a spastic paraparesis in their mid-teens. Development, speech and cognitive abilities Twenty out of 25 individuals (80%) presented with DD in early childhood. For those with epilepsy, DD preceded the onset of seizures. A global regression occurring following the onset of epilepsy was seen in three individuals. Eighteen out of 25 individuals (72%) had ID , which was borderline in three individuals, mild in nine, moderate in five and severe in one. One child had DD but was too young to make a formal diagnosis of ID. Median age at first words was 15.5 months (range: 12-36 months). At the most recent examination, eight individuals demonstrated age-appropriate speech, whereas the majority of the remaining individuals did not. Expressive language was more severely affected compared to receptive language. Median age at unsupported walking was 18 months (range 12-26 months) and all individuals were ambulant at last evaluation. Seizures and epilepsy classification Nine out of 25 (36%) had a history of seizures. In addition, in case 20 and 21 possible seizures or seizure-like events were reported, but never confirmed, and were not considered for further analysis. The most frequently reported seizure types were bilateral tonic-clonic seizures (BTCS) (cases 1, 2, 4 and 22, 25) and absence seizure (cases 5,10,17 and 25). A diagnosis of developmental and/or epileptic encephalopathy (DEE) could be made in three individuals (case s 1, 2 and 3). All had drug-resistant epilepsy, multiple seizure types and a history of status epilepticus. EEG showed focal and generalized epileptic discharges, but background was always normal. Of the remaining six, four had absences seizures, with or without BTCS while the remaining two had BTCS only. These findings are suggestive of genetic generalized epilepsy. Case 25 was diagnosed with epilepsy with eyelid myoclonia, a disorder positioned within the spectrum bridging genetic generalized epilepsy and DEE. 35 An additional three patients had an abnormal EEG but no clinical seizures. These EEG abnormalities included 3 Hz generalized spike waves in one, and centrotemporal spikes in the other two. Behavior and psychiatric features Taken together, 20 individuals (80%) had a formal or suspected neuropsychiatric disorder. ASD features were reported in 13 individuals, and attention-deficit/hyperactivity disorder ( ADHD ) features were observed in 11 individuals. Other psychiatric disorders were reported in 17 out of 25 individuals (68%), including oppositional defian t disorder, attachment disorder, Gilles de La Tourette syndrome , bipolar disorder, anxiety disorder, schizophrenia and substance abuse. Gastro-intestinal features Failure to thrive was reported in 13/25 individuals (52%). Intervention with enteral nutrition (either through gastrostomy or nasogastric tube) after the neonatal period was needed in five individuals. At last follow-up, seven of the 25 individuals (2!%) were underweight, but none of them needed enteral nutrition. Gastro-esophageal reflux disease (GERD) was reported in 10/25 individuals (40%). Three individuals had pancreatic features , ranging from pancreatitis to pancreatic insufficiency. Other co morbidities Sleep disturbances were reported in 8/25 (32%), encompassing impairments in both sleep onset and sleep maintenance. Renal abnormalities were seen in four cases: ultrasound showed nephrolithiasis in case 2. Case 13 had an episode of post-infectious nephritis but recovered completely. Case 20 was diagnosed with a medullary sponge kidney and nephrocalcinosis. Case 22 had a severe nephrotic syndrome, which led to kidney failure and dialysis. Case 24 had congenital hydronephrosis without clinical symptoms. No immunodeficiency disorders were seen, but three cases had recurrent infections of the ears or skin. None of the individuals had glycemic disturbances or diabetes. Refractive and ocular alignment disorders were seen in five individuals. Brain MRI was performed in 16/25 (64%) individuals, none of which revealed any clinically relevant abnormalities. Altered locomotor activity in cmip +/- and cmip -/- zebrafish larvae To support the pathogenicity of CMIP variants and their implications in neurodevelopmental and neuropsychiatric disorders, a zebrafish cmip mutant model was characterized. The functional impact of cmip loss on locomotor behavior was evaluated by measuring the total distance moved over 60 minutes in cmip +/+ , cmip +/- and cmip -/- zebrafish larvae at 28°C (standard temperature) (Fig. 3A-D) and 32°C (hyperthermia) (Fig. 3E-H). At 28°C, cmip +/+ , larvae demonstrate a gradual decline in locomotor activity for 40 minutes whereafter the behavior stabilizes (Fig. 3A). This can be considered as the habituation period wherein the larvae adapt to the dark condition. In contrast, cmip +/- and cmip -/- larvae habituated faster, reaching stable locomotor activity after 30 minutes (Fig. 3A). During the initial 0-20 minute period, no significant differences were observed in the locomotor activity between the different genotypes (Fig. 3B). Interestingly, during the 20-40-minute interval, both cmip +/- and cmip -/- larvae showed significantly reduced locomotor activity compared to cmip +/+ (Fig. 3C), likely reflecting their earlier habituation. After habituation, during the 40-60 minutes time interval, a non-significant increase of 23.9% was observed in the total distance moved of cmip -/- larvae compared to cmip +/+ larvae. No such trend was observed for the cmip +/- larvae during this time interval (Fig. 3D). At 32°C, cmip +/+ larvae showed a habituation period of about 40 minutes, while cmip +/- and cmip -/- larvae again seem to habituate faster in about 30 minutes (Fig. 3E). Although no significant genotype-dependent differences were apparent in the first 20 minutes (Fig. 3F), a significant decrease in locomotor activity was also observed in the 20-40 minutes time interval for cmip +/- and cmip -/- larvae compared to cmip +/+ larvae (Fig. 3G). This significant decrease in behavior persisted in the 40-60 minutes time interval for both the cmip +/- and the cmip -/- in the 40-60 minutes interval, which sharply contrasts the results at 28°C that showed an apparent increase in locomotor activity for the cmip -/- larvae (Fig. 2H). In addition, the cumulative duration spent in a highly active state was characterized, which closely aligned with the outcomes of total distance moved , demonstrating shorter habituation time and altered locomotor activity (Fig. S1). Together, these results suggest that loss of cmip function accelerates habituation to a changing environment (i.e., light – dark conditions), and alters locomotor activity in function of temperature, which persisted over time as hypoactivity under mild hyperthermic conditions. Spontaneous epileptiform activity in cmip +/- zebrafish larvae To investigate whether similar neural hyperexcitablity is present in the cmip zebrafish model, non-invasive LFP recordings were performed to characterize the presence of spontaneous epileptiform brain activity. A significant increase in the frequency and mean cumulative duration of spontaneous epileptiform events was observed for the cmip +/- larvae, butnot for cmip -/- larvae. (Fig. 4A-4B). Representative 10-minute non-invasive LFP recordings for each genotype are provided in the supplementary material (Fig. S2). During 10-minute non-invasive LFP recordings, cmip +/+ and cmip -/- larvae predominantly exhibited one to two spontaneous epileptiform-like events, whereas cmip +/- larvae frequently displayed three or more events (Fig. 4C). Among cmip +/+ larvae, none showed three or more epileptiform-like events. Furthermore, 10/20 (50%) cmip -/- larvae had at least one epileptiform event, of which only 30% experienced 3 or more events. Conversely, of the 17/23 (74%) cmip +/- larvae that had at least one epileptiform event, 76% experienced 3 or more events. Taken together, these results indicate that cmip +/- larvae demonstrate spontaneous epileptiform events. Cmip mRNA and protein expression are reduced in cmip -/- larvae To validate the cmip zebrafish model, cmip mRNA and protein expression levels were analyzed in cmip +/+ , cmip +/- and cmip -/- larvae. Quantitative RT-PCR was performed to measure relative cmip mRNA expression levels, normalized to the housekeeping genes Ube2a, Rpl13a and Hatn10 (Fig. 5A). C mip mRNA expression was robustly expressed in cmip +/+ larvae and significantly decreased by approximately 50% in cmip -/- larvae compared to cmip +/+ (Fig. 5A). Cmip +/- larvae showed a slight non-significant reduction in RNA expression levels. Western blot analysis using a C-terminal-specific antibody (Mybiosource) for canonical CMIP (86 kDa) were in line with these transcript levels (Fig. 5B). Relative CMIP protein expression normalized to GAPDH revealed abundant protein expression in cmip +/+ larvae, a significant reduction in cmip -/- larvae and a slight but non-significant reduction in cmip +/- larvae (Fig. 5C). These findings demonstrate a substantial decrease of cmip expression, at both the transcript and protein levels in cmip -/- larvae, confirming the efficacy of the allele in disrupting gene function. Discussion This study establishes a clear causal link between CMIP and neurodevelopmental and neuropsychiatric disorders. We identified 25 individuals with a clinical phenotype characterized by a high prevalence of DD/ID, ASD traits, ADHD traits, other psychiatric disorders, and drug resistant epilepsy. These clinical observations are in line with the phenotype observed in the cmip mutant zebrafish model, which demonstrated (temperature-dependent) alterations in locomotor activity in cmip ⁺/⁻ and cmip −/− larvae and spontaneous epileptiform activity in cmip +/⁻ larvae. Clinical and genetic findings We report 25 individuals with NDD linked to CMIP , including six individuals with a SNV. Despite phenotypic heterogeneity, developmental delay (20/25, 80%), intellectual disability (18/25, 72%) and neuropsychiatric features (20/25, 80%) were highly prevalent. Surprisingly, no clear differences in clinical presentation were observed between individuals harbouring single gene mutations and those with large deletions in our cohort. Neurological examination was abnormal in half of the cohort, characterised by movement disorders, including postural tremor, myoclonus and dyskinesia, but also nonspecific findings such as balance disturbances, clumsiness and mild hypotonia. Intellectual functioning ranged from normal to severe ID, likely reflecting variable expressivity, genetic background, and environmental influences, as seen in other NDD-associated genes. 31 The high prevalence of psychiatric comorbidities in CMIP is remarkable, compared to other individuals with NDD, as illustrated by internalising or externalising symptoms in up to 61% of school-age children with NDD. 32 When considering a specific genetic etiology, the high rate of psychiatric comorbidities observed in our cohort parallels findings in fragile X syndrome, where anxiety and anxiety disorders are reported in up to 86% of individuals and mood instability and aggression occur in up to 58% of individuals. 33 The involvement of CMIP in the pathophysiology of psychiatric disorders is further supported by previously reported associations of CMIP polymorphisms and schizophrenia. 34 It could be that other factors, both environmental as well as genetic (other than CMIP ), contribute to the high prevalence of psychiatric disorders in our cohort. 35 , 36 Epilepsy, although not universal, was another prominent feature in nine individuals (36%). It is striking that in cases 2 and 3, in which epilepsy was most severe, a regression was reported in all domains, after the onset of their seizures, as often is seen in other DEE. 30 The remaining individuals had absence seizures or BTCS, which were most likely genetic generalized epilepsies, supported by generalized epileptic abnormalities on EEG in some. The high prevalence of gastro-intestinal manifestations observed in this cohort aligns with prior findings in children with ASD, where the occurrence of gastro-intestinal disorders is over four-fold increased higher compared to individuals without ASD. 37 , 38 Because of the high frequency of failure to thrive in this cohort, clinicians should be aware of this potential comorbidity in CMIP -related NDD and regular follow-up of weight and dietary habits is advised. Collectively, the broad phenotypical spectrum of neurological and neuropsychiatric features of CMIP is in parallel with other neurologic disorders linked to adaptor proteins, in which the clinical picture also includes abnormal gait, spasticity, hypotonia and muscle weakness. 39 No alternative pathogenic drivers beyond CMIP Among individuals with CNVs spanning CMIP , several additional protein coding genes were co-deleted. To evaluate their potential contribution, we systematically reviewed each gene (Table S3 ). A total of ten disease-associated genes were identified (BCO1, DNAAF1, GAN, GCSH, MAF, MBTPS1, MLYCD, PLCG2, SLC38A8, WWOX ), of which only BCO1, MAF, and PLCG2 are autosomal dominant. The remainder are autosomal recessive disorders. Nearly all these genes lacked a known association with neurodevelopmental and neuropsychiatric phenotypes. Although MAF is associated with intellectual disability in Ayme-Gripp syndrome, the absence of congenital cataracts and hearing loss in the present cohort makes MAF involvement unlikely. 40 GAN causes giant axonal neuropathy, which may include ID with disease progression, but none of the individuals in this cohort had a phenotype related to it. 41 WWOX , is associated with developmental and epileptic encephalopathy and has been proposed to be a weak ASD risk factor in heterozygous individuals. 42 Since GAN and WWOX are autosomal recessive, the heterozygous deletions in our cohort are unlikely to be pathogenic. Taken together in our cohort, we found no alternative pathogenic variants, and the few co-deleted genes identified showed only limited or indirect evidence of pathogenicity. This absence of other genetic drivers strengthens the genotype–phenotype correlation, indicating that CMIP disruption is the main driver of the neurodevelopmental and neuropsychiatric features irrespective of the size of the deletion. Insights from the cmip mutant zebrafish model To explore the functional consequences of CMIP loss of function and to support the pathogenicity of human CMIP variants, a zebrafish cmip mutant model was characterized. Mutant larvae displayed altered locomotor activity and spontaneous epileptiform brain activity, paralleling patient observations. The accelerated habituation observed in cmip +/− and cmip −/− may result from altered neural circuit function due to cmip loss, a developmental delay in environmental adaptation, or underlying hypotonia as also observed in CMIP patients. The subsequent observed hypoactivity has already been reported in several genetic zebrafish epilepsy models. 27 Remarkably, the presence of spontaneous epileptiform events was only observed in cmip +/− , suggestive of heterozygous, rather than homozygous, loss of cmip predisposing to neural hyperexcitability, which mirrors the clinical observations. Taken together, the zebrafish data provide functional evidence that cmip disruption causes behavioral hypoactivity and increased seizure susceptibility, recapitulating key features of CMIP -associated NDDs. Clinically, only a subset of CMIP patients first presented with epilepsy. Although epilepsy is not the most prominent feature of a CMIP -related NDD, clinicians should be aware that different seizure types may occur, that seizures can be difficult to treat and may be accompanied with developmental regression. Notably, cmip expression was markedly reduced at the RNA and protein level in cmip −/− larvae, although the latter was not completely abolished. The cmip gene is not annotated as duplicated in zebrafish, and no alternative isoforms have been documented to account for residual expression. However, stopcodon readthrough has been reported in zebrafish, which may explain the unexpectedly high protein levels in the cmip −/− larvae. 43 The role of CMIP in neurodevelopment and epilepsy The exact pathophysiological function of CMIP in neurodevelopment has yet to be fully elucidated. However, its importance as an adaptor protein involved in neuronal migration, cytoskeletal remodelling and synaptic formation, particularly via interactions with binding partners such as filamin A and the PI3K/Akt/mTOR pathway, emphasizes the central role of CMIP in neurodevelopmental processes. 2 , 7 , 12 , 44 Future research would benefit from focusing on which specific pathways are involved in the pathophysiology of CMIP . Limitations Clinical data were collected retrospectively, and complete information was not available for all individuals. Despite these missing data, we were able to present a robust overview of the clinical phenotype. It is important to acknowledge that the relatively limited cohort size precluded a detailed analysis of genotype-phenotype correlations. In the present study, we included only individuals with deletions or nonsense variants; however, missense variants have also been reported in DECIPHER, GeneMatcher and once in a large cohort of children with neurodevelopmental disorders. 45 Some of these exhibit phenotypes similar to those observed in our cohort. Future research should therefore also address the potential pathogenicity of these variants, ideally including functional in vitro studies. Conclusion In conclusion, our findings establish CMIP as a strong candidate gene for NDD. Thus. we suggest considering CMIP as a cause for DD-related phenotypes, associated with a wide range of psychiatric disorders (including ASD and ADHD), epilepsy, gastrointestinal features and potentially movement disorders. Including CMIP in next generation sequencing-panels will lead to the discovery of additional individuals, allowing further insights in the phenotype, including CMIP variant (re)classification. Follow-up studies will refine the clinical spectrum of this disease and the molecular pathways leading to the pathogenicity of CMIP . Declarations Data availability The authors confirm that the data supporting the findings of this study are available within the article and its supplemental material. Competing interests The authors declare no competing interests. AD received funding from the KU Leuven Fund for Childhood Epilepsy ( https://www.leuvenkinderepilepsiecentrum.be/en ). Ethical approval All data were collected and processed in accordance with the European General Data Protection Regulation (GDPR; EU 2016/679) and Belgian national legislation. Site-specific informed consent was obtained to all participants. The study was approved by the Institutional Review Board of Antwerp University Hospital (B300201316250). Author contributions Conceptualization: MDW, MVDL, AJ, FK; MDW collected clinical data; MVDL, AD, DC and FK designed and conducted the experiments; all co-authors contributed to the writing-review and editing of the manuscript. Acknowledgements We are grateful to all the individuals and their family who cooperated in this study, as well as to their referring physicians. Jolien Huyghebeart and Claudio d’Incal for helping with the primer designs and Western blotting experiments. We thank Dr. Nathalie Van der Aa, MD for bringing the first case with a CMIP deletion to our attention. References Ollero M, Sahali D. The Enigmatic Emerging Role of the C-Maf Inducing Protein in Cancer. Diagnostics (Basel) . Apr 8 2021;11(4)doi: 10.3390/diagnostics11040666 Grimbert P, Valanciute A, Audard V, et al. Truncation of C-mip (Tc-mip), a new proximal signaling protein, induces c-maf Th2 transcription factor and cytoskeleton reorganization. J Exp Med . 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Running the Stop Sign: Readthrough of a Premature UAG Termination Signal in the Translation of a Zebrafish (Danio rerio) Taurine Biosynthetic Enzyme. Mar Drugs . Jun 3 2017;15(6)doi: 10.3390/md15060162 Oniszczuk J, Sendeyo K, Chhuon C, et al. CMIP is a negative regulator of T cell signaling. Cell Mol Immunol . Oct 2020;17(10):1026–1041. doi: 10.1038/s41423-019-0266-5 Shillington A, Lamy M, Dominick KC, Sorter M, Erickson CA, Hopkin R. A Collaborative Psychiatric-Genetics Inpatient Care Delivery Model Improves Access to Clinical Genetic Evaluation, Testing, and Diagnosis for Patients With Neurodevelopmental Disorders. Front Genet . 2022;13:901458. doi: 10.3389/fgene.2022.901458 Tables Table 1 is available in the Supplementary Files section. Additional Declarations There is no duality of interest Supplementary Files STROBEchecklistv4cohortCMIP161025.doc Strobe Checklist supplementalmaterialCMIP.pdf Supplementalmaterial CMIPsupplementalworksheet.docx Supplementalmaterial_clinicalworksheet Table1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 16 Mar, 2026 Review # 2 received at journal 04 Mar, 2026 Reviewer # 2 agreed at journal 17 Feb, 2026 Review # 1 received at journal 09 Feb, 2026 Reviewer # 1 agreed at journal 04 Feb, 2026 Reviewers invited by journal 12 Jan, 2026 Submission checks completed at journal 05 Jan, 2026 First submitted to journal 23 Dec, 2025 Unknown event 11 Dec, 2025 Editor assigned by journal 09 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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13:03:13","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24698,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineCMIPfigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/0583cbfb5cfabd551b7aa051.png"},{"id":100406604,"identity":"16a62a6a-86bf-4f8b-8261-ee0777d16f84","added_by":"auto","created_at":"2026-01-16 13:03:03","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":707141,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineCMIPfigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/8e9a4ca2a9a4275a0c4e4f07.png"},{"id":100406635,"identity":"d4bcc800-bee9-4042-9d31-2447febf858c","added_by":"auto","created_at":"2026-01-16 13:03:05","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":244608,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineCMIPfigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/0a06f1fc345766122b68d323.png"},{"id":100406758,"identity":"080b0e39-bab6-416c-86d8-1224ef98583c","added_by":"auto","created_at":"2026-01-16 13:03:16","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26750,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineCMIPfigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/58cfb8ff64a798e1a30ed836.png"},{"id":100406318,"identity":"03088332-95d8-4919-9cf9-61d789116aab","added_by":"auto","created_at":"2026-01-16 13:00:19","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":395319,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/fc9b3710e314a34fcb25a10b.png"},{"id":100406725,"identity":"5bcdedff-9478-4230-b9ae-18213e09e7c8","added_by":"auto","created_at":"2026-01-16 13:03:13","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":186930,"visible":true,"origin":"","legend":"","description":"","filename":"153925EJHG0structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/d0101cc33b27fbbec0e8a555.xml"},{"id":100406541,"identity":"3ae40c83-5a81-49b7-8f90-17d523e670b6","added_by":"auto","created_at":"2026-01-16 13:02:59","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":212940,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/e756d498648be05e7fb14e8c.html"},{"id":100406206,"identity":"97ff5ba6-e9ae-463e-aef0-3871807ec0ff","added_by":"auto","created_at":"2026-01-16 12:54:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2305902,"visible":true,"origin":"","legend":"\u003cp\u003eCharacteristics of CMIP cohort. The numbers represent the number of individuals in these categories. ADHD: attention-deficit/hyperactivity disorder traits; ASD: autism spectrum disorder traits; (*): The two individuals classified as having ‘unknown’ seizures experienced paroxysmal considered possibly epileptic but not confirmed.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/512a01dccd03888e363c751e.jpg"},{"id":100405818,"identity":"48d64b00-2046-48c1-962e-c4f0701fdfa2","added_by":"auto","created_at":"2026-01-16 12:18:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113250,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic location of CMIP and overlapping CNVs in affected individuals. UCSC Genome Browser view of chromosome 16q21–q23.1 (hg19 assembly) showing the position of CMIP and adjacent protein-coding genes (blue). Red bars indicate the genomic intervals of copy number variants (CNVs) detected in affected individuals. Overlaps across multiple cases delineate a minimal critical region encompassing \u0026nbsp;CMIP, supporting its role as a candidate gene contributing to neurodevelopmental and epileptic phenotypes.\u003c/p\u003e","description":"","filename":"CMIPfigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/87c25185eb7f3ed8bc306557.jpg"},{"id":100421860,"identity":"953c6f8c-a883-4aeb-a9a8-3058d6f5e1d2","added_by":"auto","created_at":"2026-01-16 13:57:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2769214,"visible":true,"origin":"","legend":"\u003cp\u003eLocomotor activity of cmip+/+, cmip+/- and cmip-/- zebrafish larvae at 28°C and 32°C at 7 dpf expressed as total distance moved (mm). Total distance moved of cmip+/+ (28°C: n=58-61, 32°C: n=40-41), cmip+/- (28°C: n= 65-67, 32°C: n=43-45) and cmip-/- (28°C: n=28-32, 32°C: n=34-36°C) larvae is recorded over a period of 60 min in the dark at (A-D) 28°C and (E-H) 32°C. Behavioral data is compared to cmip+/+ larvae and expressed as total distance moved (mm) (mean ± SEM). (A,E) Behavioral trajectory of cmip+/+, \u0026nbsp;cmip+/- and cmip-/- zebrafish larvae, quantified for (B,F) the 0-20 min interval, (C,G) the 20-40 min interval and the (D,H) 40-60 min interval. Statistical analysis was performed using (A,E) two-way ANOVA with Dunnett’s multiple comparison test and (B-D,F-H) one-way ANOVA with Dunnett’s multiple comparison test. Outliers were removed via the ROUT test (Q = 0.5 %) (Graphpad Prism 10, San Diego, CA, USA). Significance levels: *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"CMIPfigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/566eb489a152cf4f85a998d7.jpg"},{"id":100405400,"identity":"6b54416d-94a8-46af-8dc0-93107cb3ba7a","added_by":"auto","created_at":"2026-01-16 12:05:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1062706,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophysiological epileptiform activity of 7 dpf cmip+/+ (n = 14-21), cmip+/- (n= 17-24) and cmip-/- (n = 20-21) larvae is assessed via non-invasive local field potential recordings at room temperature. (A,B) Electrophysiological data is normalized to cmip+/+ and expressed as (A) the number of epileptiform events (ms) (mean ±SEM) and (B) cumulative duration of epileptiform events (mean ±SEM). (C) Quantification of larvae per genotype experiencing 3 or more epileptiform events. (A-B) Statistical analysis was performed using non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons test (*p\u0026lt;0.05, ***p\u0026lt;0.001 ****p\u0026lt;0.0001) and (C) Fisher’s exact test. Outliers were removed via the ROUT test (Q = 0.5) (Graphpad Prism 10, San Diego, CA, USA).\u003c/p\u003e","description":"","filename":"CMIPfigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/ba9d171ced3803f60a82895d.jpg"},{"id":100405561,"identity":"2882edf7-5db8-48a8-8d25-5dc61cfe4982","added_by":"auto","created_at":"2026-01-16 12:07:40","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":66612,"visible":true,"origin":"","legend":"\u003cp\u003eReduction in mRNA and protein expression in cmip-/- zebrafish larvae. (A) RT-PCR analysis showed a slight decrease in cmip mRNA expression in cmip+/- larvae, and a significant reduction in cmip-/- larvae compared with cmip+/+ larvae. (B) Western blotting of CMIP with C-terminal antibody at 86kDa. (C) Relative cmip protein expression normalized to Gapdh, showed a slight but non-significant decrease in cmip+/- larvae and a significant reduction in cmip-/- zebrafish larvae compared to cmip+/+. Data are presented as mean ± SEM. *p\u0026lt;0.05, ****p\u0026lt;0.0001Sample sizes: n = 3-4 per group (each sample consisting of pooled brains from 5–10 larvae.\u003c/p\u003e","description":"","filename":"CMIPfigure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/8ffc65a087c6999f0742a10d.jpg"},{"id":100551271,"identity":"79ca843a-506f-4100-8abd-3a9e8e38ab8a","added_by":"auto","created_at":"2026-01-19 08:35:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7492015,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/acc99708-7d34-4bde-a66a-ad0faae6ff9b.pdf"},{"id":100404944,"identity":"11c35332-d1a1-4df7-9495-bc6eda5e9dd1","added_by":"auto","created_at":"2026-01-16 12:04:29","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":83456,"visible":true,"origin":"","legend":"Strobe Checklist","description":"","filename":"STROBEchecklistv4cohortCMIP161025.doc","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/99b94c9d1ece1193164b96e4.doc"},{"id":100421507,"identity":"a863ef49-0e1a-495e-90c9-f0f30249ef70","added_by":"auto","created_at":"2026-01-16 13:33:10","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":829513,"visible":true,"origin":"","legend":"Supplementalmaterial","description":"","filename":"supplementalmaterialCMIP.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/d7a9a8509caa1bfd7fa3073f.pdf"},{"id":100406584,"identity":"b565a774-0d7e-424d-937b-d1681655bb59","added_by":"auto","created_at":"2026-01-16 13:03:02","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18129,"visible":true,"origin":"","legend":"Supplementalmaterial_clinicalworksheet","description":"","filename":"CMIPsupplementalworksheet.docx","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/cdf626dd2d5c5f51272b6efb.docx"},{"id":100406721,"identity":"637fb2a2-55c0-498e-8461-c72e677e0a7c","added_by":"auto","created_at":"2026-01-16 13:03:12","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":27303,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8316803/v1/2b3331bf5309f5235a03d36a.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"CMIP as a novel candidate gene for neurodevelopmental and neuropsychiatric disorders","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eCMIP\u003c/em\u003e (\u003cem\u003ec-maf inducing protein\u003c/em\u003e), located on chromosome 16q23.2, encodes a multi-domain adaptor protein composed of an N-terminal pleckstrin homology domain, a central region with multiple protein interaction motifs, and a C-terminal leucine-rich repeat domain.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e It is expressed mainly in the brain and in other tissues, including increased expression in peripheral mononuclear blood cells and kidney.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e In the central nervous system, CMIP modulates network excitability mainly by repressing cortical interneuron activity, a mechanism increasingly recognized in the pathophysiology of neurodevelopmental and neuropsychiatric disorders.\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eProtein interactors of CMIP include the RelA subunit of nuclear factor kappa-B (NF-κB) transcription factor ,\u003csup\u003e6\u003c/sup\u003e the p85 regulatory subunit of phosphoinositide-3 kinase (PI3K), and the death activated protein kinase-interacting protein 1 (Dip1),\u003csup\u003e1\u003c/sup\u003e linking CMIP to multiple signaling pathways relevant to neural development and immune function. It also interacts with filamin A, a key player in neuronal migration.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e The PI3K/Akt/mTOR pathway is a central regulator of neural progenitor proliferation, neuronal differentiation, migration, axon growth and synaptic plasticity. Disruption of this pathway during critical periods of brain development can lead to abnormal cortical architecture, imbalanced excitatory/inhibitory (E/I) signaling and impaired synaptic maturation features commonly observed in neurodevelopmental disorders (NDD) and autism spectrum disorder (ASD).\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Genome-wide association studies (GWAS) have identified \u003cem\u003eCMIP\u003c/em\u003e as a candidate gene for specific language impairment (SLI), reading abilities and general cognitive function.\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e In this context, \u003cem\u003eCMIP\u003c/em\u003e emerged as a potential candidate gene for NDD following two independent case reports, in which ASD and developmental delay (DD) were the most striking features. These individuals had a complete or partial deletion of \u003cem\u003eCMIP\u003c/em\u003e, in combination with adjacent genes at chromosome 16q23.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e The first reported case presented with global DD during infancy and febrile seizures. Over time language acquisition was more significantly impaired and the child was diagnosed with ASD.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e In the other publication, including two independent cases, a similar clinical phenotype was observed, accompanied by gastrointestinal symptoms, suggesting a syndromic form of ASD.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDespite these findings, the link between \u003cem\u003eCMIP\u003c/em\u003e and NDD and/or neuropsychiatric disorders remained a matter of debate. In this study, we outline the pathogenicity of \u003cem\u003eCMIP\u003c/em\u003e through a multicentre case series of 25 individuals with \u003cem\u003eCMIP\u003c/em\u003e-related NDD, alongside functional assessment in a \u003cem\u003eCMIP\u003c/em\u003e loss-of-function zebrafish mutant model. Together, these data define the phenotypic spectrum associated with \u003cem\u003eCMIP\u003c/em\u003e variants and support its classification as a novel NDD gene.\u003c/p\u003e"},{"header":"Materials (Subjects) and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and collection of clinical characteristics\u003c/h2\u003e \u003cp\u003eThis multicentre, retrospective observational study described clinical features of individuals with \u003cem\u003eCMIP\u003c/em\u003e-related neurodevelopmental disorder, recruited through GeneMatcher and DECIPHER between 2020 and 2025.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Data were collected using a standardized datasheet provided to participating clinicians (supplemental material). Genetic analyses were performed according to local protocols. Epilepsy was defined and classified according to the 2022 classification of the International League Against Epilepsy (ILAE).\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Developmental delay was defined as failure to meet expected developmental milestones in one or more domains. Intellectual disability (ID) was defined according to DSM-4 and DSM-5, based on formal intelligence coefficient testing,if available (normal: \u0026ge; 85, borderline: 70\u0026ndash;84; mild 55\u0026ndash;69; moderate 40\u0026ndash;54; severe or profound\u0026thinsp;\u0026lt;\u0026thinsp;39).\u003csup\u003e22,23\u003c/sup\u003e When testing was unavailable, classification relied on clinical records and required support level.The manuscript followed STROBE guidelines supplemental material).\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReview of the literature\u003c/h3\u003e\n\u003cp\u003eTo outline the phenotypical spectrum of individuals with a \u003cem\u003eCMIP\u003c/em\u003e-related NDD, a PubMed literature search (September 2025) was performed using the terms: (CMIP) AND (\"neurodevelopmental disorders\"[MeSH Terms] OR (neurodevelop* disorder*[Tiab]) OR (\"child mental disorder*\"[Tiab]) OR (\"intellectual disab*\"[Tiab]) OR (\"developmental delay\"[Tiab])), identifying eight articles and two additional individuals [22].\u003c/p\u003e\n\u003ch3\u003eZebrafish model and husbandry\u003c/h3\u003e\n\u003cp\u003eHeterozygous \u003cem\u003ecmip\u003c/em\u003e zebrafish (sa43067), carrying a premature stop codon (g.13351012A\u0026thinsp;\u0026gt;\u0026thinsp;T), were obtained from the European Zebrafish Resource Center and originally generated by ENU mutagenesis at the Stemple Lab.\u003c/p\u003e \u003cp\u003eAdult fish were maintained at 28.5\u0026deg;C in UV-sterilized water on a 14/10h light/dark cycle under standard aquaculture conditions. Fertilized embryos were collected via natural spawning and raised at 28\u0026deg;C in embryo medium (1.5 mM HEPES, pH 7.2, 17 mM NaCl, 2 mM KCl, 0.12 mM MgSO4, 1.8 mM Ca(NO3)2, 0.6 \u0026micro;M methylene blue).\u003c/p\u003e\n\u003ch3\u003eBehavioral analysis\u003c/h3\u003e\n\u003cp\u003eCmip zebrafish larvae (7 dpf) were transferred individually into 96-well plates containing 100 \u0026micro;l Danieau\u0026rsquo;s medium, and behavior was monitored for 60 minutes in darkness at 28\u0026deg;C or 32\u0026deg;C using DanioVision\u0026trade; (Noldus, The Netherlands).\u003c/p\u003e \u003cp\u003eThe behavioral metrics are calculated for every 5-min period. Ethovision XT16 (Noldus, Netherlands) was used to quantify locomotor behavior as total distance moved (mm) per 5 min and as cumulative duration in highly active state (s) per 5 min Activity was defined as pixel changes between consecutive images, ranging from 0% (no change) to 100% (all pixels changed). The highly active state has previously been associated with seizure-like behavior.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eNon-invasive local field potential (LFP) recordings\u003c/h3\u003e\n\u003cp\u003eNon-invasive LFP recordings of 7 dpf larvae were performed as previously described.\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Signals were recorded from the optic tectum for 10 minutes at room temperature using glass electrodes filled with artificial cerebrospinal fluid (124 mM NaCl, 2 mM KCl, 2 mM MgSO4, 2 mM CaCl2, 1.25 mM KH2PO4, 26 mM NaHCO3 and 10 mM glucose). Epileptiform events were defined as discharges\u0026thinsp;\u0026ge;\u0026thinsp;threefold baseline amplitude and \u0026ge;\u0026thinsp;50 ms duration and quantified using Clampfit 10.2 (Molecular Devices Corporation, USA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGenotyping\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from 7 dpf larvae andlysed with 50 mM NaOH at 95\u0026deg;C for 10 minutes and neutralized with Tris-HCl (pH 8.0). Genotyping PCR was performed using GoTaq\u0026reg; G2 DNA Polymerase (Promega) with custom primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) at 58\u0026deg;C. Amplicons were analyzed by QIAgel capillary electrophoresis and Sanger sequencing and aligned to the \u003cem\u003ecmip\u003c/em\u003e reference genome using CLC Genomics Workbench 5.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA extraction and RT-PCR\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eRNA extraction and RT-PCR\u003c/div\u003e \u003cp\u003eRNA was extracted from pooled larval brains (n\u0026thinsp;=\u0026thinsp;5\u0026ndash;10 per genotype) using the RNeasy Micro Kit (Qiagen). RNA concentration and purity were measured using Qubit\u0026trade; RNA assay (Invitrogen\u0026trade;) and NanoDrop\u0026trade; Spectrophotometer (Thermo Scientific).\u003c/p\u003e \u003cp\u003eFor RT-PCR, 1 \u0026micro;g of RNA was reverse-transcribed using the Superscript III First-Strand Synthesis System (Invitrogen) and primer efficiencies were optimized using standard dilution curves. RT-PCR was performed in triplicate with SYBR Green I Mastermix (Eurogentec) on a CFX384 Touch system (BioRad, USA), with primers listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Data were analyzed in in QBASE+ (Biogazelle, Belgium), using \u003cem\u003eUbe2a, Rpl3\u003c/em\u003e and \u003cem\u003eHatn10\u003c/em\u003e as reference genes.\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eProtein was isolated from pooled zebrafish larvae brains (n\u0026thinsp;=\u0026thinsp;5\u0026ndash;10 per genotype) and homogenized using a TissueRuptor II (Qiagen) in ice cold RIPA buffer (150 mM NaCl, 50 mM Tris, 0.5% sodium deoxycholate, 1% NP-40 and 2% SDS) supplemented with protease (cOmplete\u0026trade; Mini, Roche) and phosphatase (PhosSTOP\u0026trade;, Roche) inhibitors. Lysates were incubated on ice for 15 minutes and cleared by centrifugation for 10 minutes at maximal speed. Protein concentration was determined with the Pierce\u0026trade; BCA Protein Assay Kit (Thermo Scientific).\u003c/p\u003e \u003cp\u003eEqual amounts of protein (7\u0026micro;g) were mixed with NuPAGE\u0026trade; Sample Reducing Agent (Invitrogen) in Laemmli buffer (Bio-Rad), heated at 70\u0026deg;C for 10 min, and separated on Bolt\u0026trade; 4\u0026ndash;12% Bis-Tris gels (Invitrogen) in MOPS buffer. Proteins were transferred onto nitrocellulose membranes (Cytiva) using a Mini Trans-Blot\u0026reg; Cell (Bio-Rad). Transfer efficiency was verified by Ponceau S staining (Sigma). Membranes were blocked in 5% non-fat dry milk (Carl Roth) in TBST for 1 hour at room temperature, followed by overnight incubation at 4\u0026deg;C with primary CMIP antibody (Proteintech 12851-1-AP, UK; 1/1000. HRP-conjugated immunoglobulins (Agilent Santa Clara,, USA; 1/2000) were appliedunder identical conditions. Signals were detected using SuperSignal\u0026trade; West Pico PLUS (Thermo Scientific),. with West Femto used for CMIP to enhance sensitivity Images were acquired with an Amersham\u0026trade; Imager800 (Cytiva, USA), using GAPDH (Cell signaling technology, USA) as loading control and quantifiedusing ImageJ/Fiji software.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eClinical data were analysed using IBM SPSS Statistics software, version 29 (IBM Corp., Armonk, NY, USA). Normally distributed values were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, non-normally distributed values were represented as median (Q1-Q3). median with quartile 1 (Q1) and 3 (Q3). Zebrafish dataare presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analyses were performed using one- or two-way ANOVA with with Dunnett\u0026rsquo;s post hoc test or Kruskal\u0026ndash;Wallis test with Dunn\u0026rsquo;s multiple comparisons. Outliers were identified via the ROUT test (Q\u0026thinsp;=\u0026thinsp;0.5%) and excludedin GraphPad Prism 10 (GraphPad Software, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eClinical and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emolecular characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe identified 22 previously unreported individuals with \u003cem\u003eCMIP\u003c/em\u003e-related neurodevelopmental disorders and updated clinical and genetic data for three previously published cases, including one reported by our group (Table 1). Median age at last evaluation was 15 years (range: 1.3-64 years) 14 individuals were male and 11 were female. Altogether, these 25 individuals originated from 18 unrelated families.\u003c/p\u003e\n\u003cp\u003eSix different truncating single nucleotide variants (SNVs) were present in seven of the 25 individuals (28%). The remaining 18 individuals (72%) had a complete or partial deletion of \u003cem\u003eCMIP\u003c/em\u003e, which also included additional protein coding genes (Fig. 2). These deletions ranged between 283 kb and 6,97 Mb. In 12/18 cases (67%), the entire \u003cem\u003eCMIP\u003c/em\u003e gene was deleted. Additional protein coding genes included in the deletions are: \u003cem\u003eADAD2, ATMIN, ATP2C2, BCO1, C16orf46, CDH13, CDYL2, CENPN, CIBAR2,\u0026nbsp;\u003c/em\u003eCLEC3A, \u003cem\u003eCMC2, COTL1, CRISPLD2, DNAAF1, DYNLRB2, GAN, GCSH, GSE1, HSBP1, HSD17B2, HSDL1, KCNG4, KIAA0513, KLHL36, MAF, MBTPBS1, MEAK7, MLYCD, MPHOSPH6, NECAB2, OSGIN1, PKD1L2, PLCG2, SDR42E1, SCL38A8, TAF1C, USP10, WFDC1, WWOX\u0026nbsp;\u003c/em\u003eand \u003cem\u003eZDHHC7\u003c/em\u003e, based on genomic coordinates (hg19: chr16:77,983,154 - 85,673,761), spanned from the most proximal to the most distal. A detailed overview of the \u003cem\u003eCMIP\u003c/em\u003e deletions and the affected protein coding genes is provided in Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePerinatal history\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTen out of 25 individuals (40%) were born premature, between 26 and 37 weeks of gestational age. Birth biometry was unremarkable, apart from case\u003cspan lang=\"EN-GB\"\u003es\u003c/span\u003e 20 and 21 who were large for gestational age, and case 24 who was small for gestational age. Neonatal \u003cspan lang=\"EN-GB\"\u003emanifestations\u003c/span\u003e included respiratory or feeding \u003cspan lang=\"EN-GB\"\u003efeatures\u003c/span\u003e, but all individuals were discharged without major complications. Case 10 underwent surgical intervention to repair a complex congenital heart defect. \u0026nbsp;In 4/25 (16%) individuals hypotonia in the perinatal period was reported.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical examination\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDysmorphic facial features were reported in 8/25 (33%) of individuals. Macrocephaly was observed in three individuals\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eFrontal bossing, a broad forehead, and a prominent nose were reported in multiple families. However, no distinctive dysmorphic features could be identified. Variable abnormalities including balance \u003cspan lang=\"EN-GB\"\u003eabnormalities\u003c/span\u003e and movement disorders were seen at neurological examination in 12 out of 25 individuals (48%). Two individuals from the same family (family 4) developed a spastic paraparesis in their mid-teens.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDevelopment, speech and cognitive abilities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty out of 25 individuals (80%) presented with DD in early childhood. For those with epilepsy, DD preceded the onset of seizures. A global regression occurring following the onset of epilepsy was seen in three individuals. Eighteen out of 25 individuals (72%) had \u003cspan lang=\"EN-GB\"\u003eID\u003c/span\u003e, which was borderline in three individuals, mild in nine, moderate in five and severe in one. One child had DD but was too young to make a formal diagnosis of ID. Median age at first words was 15.5 months (range: 12-36 months). At the most recent examination, eight individuals demonstrated age-appropriate speech, whereas the majority of the remaining individuals did not. Expressive language was more severely affected compared to receptive language. Median age at unsupported walking was 18 months (range 12-26 months) and all individuals were ambulant at last evaluation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeizures and epilepsy classification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNine out of 25 (36%) had a history of seizures. In addition, in case 20 and 21 possible seizures or seizure-like events were reported, but never confirmed, and were not considered for further analysis. The most frequently reported seizure types were bilateral tonic-clonic seizures (BTCS) (cases 1, 2, 4 and 22, 25) and absence seizure (cases 5,10,17 and 25). A diagnosis of developmental \u003cspan lang=\"EN-GB\"\u003eand/or\u0026nbsp;\u003c/span\u003e epileptic encephalopathy (DEE) could be made in three individuals (case\u003cspan lang=\"EN-GB\"\u003es\u003c/span\u003e 1, 2 and 3). All \u003cspan lang=\"EN-GB\"\u003ehad drug-resistant\u003c/span\u003e epilepsy, multiple seizure types and a history of status epilepticus. EEG showed focal and generalized epileptic discharges, but background was always normal. Of the remaining six, four had absences seizures, with or without BTCS while the remaining two had BTCS only. These findings are suggestive of genetic generalized epilepsy. Case 25 was diagnosed with epilepsy with eyelid myoclonia, a disorder positioned within the spectrum bridging genetic generalized epilepsy and DEE.\u003csup\u003e35\u003c/sup\u003e An additional three patients had an abnormal EEG but no clinical seizures. These EEG abnormalities included 3 Hz generalized spike waves in one, and centrotemporal spikes in the other two.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavior and psychiatric\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;features\u003c/strong\u003e\u003cspan lang=\"EN-GB\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eTaken together, 20 individuals (80%) had a formal or suspected neuropsychiatric disorder. ASD features were reported in 13 individuals, and \u003cspan lang=\"EN-GB\"\u003eattention-deficit/hyperactivity disorder (\u003c/span\u003eADHD\u003cspan lang=\"EN-GB\"\u003e)\u003c/span\u003e features were observed in 11 individuals. \u003cspan lang=\"EN-GB\"\u003eOther psychiatric disorders were reported in 17 out of 25 individuals (68%),\u003c/span\u003e including oppositional defian\u003cspan lang=\"EN-GB\"\u003et\u003c/span\u003e disorder, attachment disorder, Gilles de La Tourette\u003cspan lang=\"EN-GB\"\u003e\u0026nbsp;syndrome\u003c/span\u003e, bipolar disorder, anxiety disorder, schizophrenia and substance abuse.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGastro-intestinal\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003efeatures\u003c/strong\u003e\u003cspan lang=\"EN-GB\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eFailure to thrive was reported in 13/25 individuals (52%). Intervention with enteral nutrition (either through gastrostomy or nasogastric tube) after the neonatal period was needed in five individuals. At last follow-up, seven of the 25 individuals (2!%) were underweight, but none of them needed enteral nutrition. Gastro-esophageal reflux disease (GERD) was reported in 10/25 individuals (40%). Three individuals had pancreatic \u003cspan lang=\"EN-GB\"\u003efeatures\u003c/span\u003e, ranging from pancreatitis to pancreatic insufficiency.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOther co\u003c/strong\u003e\u003cstrong\u003emorbidities\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSleep disturbances were reported in 8/25 (32%), encompassing impairments in both sleep onset and sleep maintenance. \u003cspan lang=\"EN-GB\"\u003eRenal abnormalities\u003c/span\u003e were seen in four cases: ultrasound showed nephrolithiasis in case 2. Case 13 had an episode of post-infectious nephritis but recovered completely. Case 20 was diagnosed with a medullary sponge kidney and nephrocalcinosis. Case 22 had a severe nephrotic syndrome, which led to kidney failure and dialysis. Case 24 had congenital hydronephrosis without clinical symptoms. No immunodeficiency disorders were seen, but three cases had recurrent infections of the ears or skin. None of the individuals had glycemic disturbances or diabetes. Refractive and ocular alignment disorders were seen in five individuals. Brain MRI was performed in 16/25 (64%) individuals, \u0026nbsp;none of which revealed any clinically relevant abnormalities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Altered locomotor activity in \u003cem\u003ecmip\u003csup\u003e+/-\u003c/sup\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;cmip\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish larvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo support the pathogenicity of \u003cem\u003eCMIP\u003c/em\u003e variants and their implications in neurodevelopmental and neuropsychiatric disorders, a zebrafish \u003cem\u003ecmip\u003c/em\u003e mutant model was characterized. The functional impact of \u003cem\u003ecmip\u003c/em\u003e loss on locomotor behavior was evaluated by measuring the total distance moved over 60 minutes in \u003cem\u003ecmip\u003csup\u003e+/+\u003c/sup\u003e, cmip\u003csup\u003e+/-\u003c/sup\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;cmip\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e zebrafish larvae at 28\u0026deg;C (standard temperature) (Fig. 3A-D) and 32\u0026deg;C (hyperthermia) (Fig. 3E-H). At 28\u0026deg;C, \u003cem\u003ecmip\u003csup\u003e+/+\u003c/sup\u003e,\u0026nbsp;\u003c/em\u003elarvae demonstrate a gradual decline in locomotor activity for 40 minutes\u003cspan lang=\"EN-GB\"\u003e\u0026nbsp;whereafter the behavior stabilizes\u0026nbsp;\u003c/span\u003e(Fig. 3A). \u003cspan lang=\"EN-GB\"\u003eThis can be considered as the habituation period wherein the larvae adapt to the dark condition.\u0026nbsp;\u003c/span\u003eIn contrast, \u003cem\u003ecmip\u003csup\u003e+/-\u003c/sup\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;cmip\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e larvae habituated faster, reaching stable locomotor activity after 30 minutes (Fig. 3A). During the initial 0-20 minute period, no significant differences were observed in the locomotor activity between the different genotypes (Fig. 3B). Interestingly, during the 20-40-minute interval, both \u003cem\u003ecmip\u003csup\u003e+/-\u003c/sup\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;cmip\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e larvae showed significantly reduced locomotor activity compared to \u003cem\u003ecmip\u003csup\u003e+/+\u003c/sup\u003e\u003c/em\u003e (Fig. 3C), likely reflecting their earlier habituation. After habituation, during the 40-60 minutes time interval, a non-significant increase of 23.9% was observed in the total distance moved of \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e larvae compared to \u003cem\u003ecmip\u003csup\u003e+/+\u003c/sup\u003e\u003c/em\u003e larvae. No such trend was observed for the \u003cem\u003ecmip\u003csup\u003e+/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003elarvae during this time interval (Fig. 3D).\u003c/p\u003e\n\u003cp\u003e\u003cspan lang=\"EN-GB\"\u003eAt 32\u0026deg;C,\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan lang=\"EN-GB\"\u003ecmip\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003csup\u003e\u003cspan lang=\"EN-GB\"\u003e+/+\u0026nbsp;\u003c/span\u003e\u003c/sup\u003e\u003c/em\u003e\u003cspan lang=\"EN-GB\"\u003elarvae showed a habituation period of about 40 minutes, while\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan lang=\"EN-GB\"\u003ecmip\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003csup\u003e\u003cspan lang=\"EN-GB\"\u003e+/-\u003c/span\u003e\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cspan lang=\"EN-GB\"\u003eand\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan lang=\"EN-GB\"\u003ecmip\u003c/span\u003e\u003c/em\u003e\u003cem\u003e\u003csup\u003e\u003cspan lang=\"EN-GB\"\u003e-/-\u003c/span\u003e\u003c/sup\u003e\u003c/em\u003e\u003cspan lang=\"EN-GB\"\u003e\u0026nbsp;larvae again seem to habituate faster in about 30 minutes\u0026nbsp;\u003c/span\u003e(Fig. 3E). Although no significant genotype-dependent differences were apparent in the first 20 minutes (Fig. 3F), a significant decrease in locomotor activity was also observed in the 20-40 minutes time interval for \u003cem\u003ecmip\u003csup\u003e+/-\u003c/sup\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003ecmip\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e larvae compared to \u003cem\u003ecmip\u003csup\u003e+/+\u003c/sup\u003e\u003c/em\u003e larvae (Fig. 3G). This significant decrease in behavior persisted in the 40-60 minutes time interval for both the \u003cem\u003ecmip\u003csup\u003e+/-\u003c/sup\u003e\u003c/em\u003e and the \u003cem\u003ecmip\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e in the 40-60 minutes interval, which sharply contrasts the results at 28\u0026deg;C \u0026nbsp;that showed an apparent increase in locomotor activity for the \u003cem\u003ecmip\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e larvae (Fig. 2H). In addition, the cumulative duration spent in a highly active state was characterized, which closely aligned with the outcomes of total distance moved\u003cspan lang=\"EN-GB\"\u003e, demonstrating\u003c/span\u003e shorter habituation time and altered locomotor activity (Fig. S1).\u0026nbsp;Together, these results suggest that loss of \u003cem\u003ecmip\u003c/em\u003e function accelerates habituation to a changing environment (i.e., light \u0026ndash; dark conditions), and alters locomotor activity in function of temperature, which persisted over time as hypoactivity under mild hyperthermic conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpontaneous epileptiform activity in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e zebrafish larvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether similar neural hyperexcitablity is present in the \u003cem\u003ecmip\u0026nbsp;\u003c/em\u003ezebrafish model, non-invasive LFP recordings were performed to characterize the presence of spontaneous epileptiform brain activity. A significant increase in the frequency and mean cumulative duration of spontaneous epileptiform events was observed for the \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e larvae, butnot for \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003elarvae. (Fig. 4A-4B). Representative 10-minute non-invasive LFP recordings for each genotype are provided in the supplementary material (Fig. S2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDuring 10-minute non-invasive LFP recordings, \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e and\u0026nbsp;\u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003elarvae predominantly exhibited one to two spontaneous epileptiform-like events, whereas\u0026nbsp;\u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e larvae frequently displayed three or more events (Fig. 4C). Among \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e larvae, none showed three or more epileptiform-like events. Furthermore, 10/20 (50%) \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003elarvae had at least one epileptiform event, of which only 30% experienced 3 or more events. Conversely, of the 17/23 (74%) \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e larvae that had at least one epileptiform event, 76% experienced 3 or more events. Taken together, these results indicate that\u0026nbsp;\u003cem\u003e\u003cspan lang=\"EN-GB\"\u003ecmip\u003c/span\u003e\u003c/em\u003e\u003csup\u003e\u003cspan lang=\"EN-GB\"\u003e+/-\u0026nbsp;\u003c/span\u003e\u003c/sup\u003e\u003cspan lang=\"EN-GB\"\u003elarvae demonstrate\u0026nbsp;\u003c/span\u003espontaneous epileptiform events.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCmip\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003emRNA and protein expression are reduced in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e larvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the \u003cem\u003ecmip\u003c/em\u003e zebrafish model, \u003cem\u003ecmip\u0026nbsp;\u003c/em\u003emRNA and protein expression levels were analyzed in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e, \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e and \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e larvae. Quantitative RT-PCR was performed to measure relative \u003cem\u003ecmip\u0026nbsp;\u003c/em\u003emRNA expression levels, normalized to the housekeeping genes \u003cem\u003eUbe2a, Rpl13a\u0026nbsp;\u003c/em\u003eand \u003cem\u003eHatn10\u0026nbsp;\u003c/em\u003e(Fig. 5A). \u003cem\u003e\u003cspan lang=\"EN-GB\"\u003eC\u003c/span\u003e\u003c/em\u003e\u003cem\u003emip\u003c/em\u003e mRNA expression was robustly expressed in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e larvae and significantly decreased by approximately 50% in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e larvae compared to \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e (Fig. 5A). \u003cem\u003eCmip\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e larvae showed a slight non-significant reduction in RNA expression levels.\u003c/p\u003e\n\u003cp\u003eWestern blot analysis using a C-terminal-specific antibody\u003cspan lang=\"EN-GB\"\u003e\u0026nbsp;(Mybiosource)\u003c/span\u003e for canonical CMIP\u003cspan lang=\"EN-GB\"\u003e\u0026nbsp;\u003c/span\u003e (86 kDa) were in line with these transcript levels (Fig. 5B). Relative \u003cspan lang=\"EN-GB\"\u003eCMIP\u003c/span\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eprotein expression normalized to GAPDH revealed abundant protein expression in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/+\u0026nbsp;\u003c/sup\u003elarvae, a significant reduction in\u003cem\u003e\u0026nbsp;cmip\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e larvae and a slight but non-significant reduction in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/-\u003c/sup\u003e larvae (Fig. 5C). These findings demonstrate a substantial decrease of \u003cem\u003ecmip\u003c/em\u003e expression, at both the transcript and protein levels in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003elarvae, confirming the efficacy of the allele in disrupting gene function.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study establishes a clear causal link between \u003cem\u003eCMIP\u003c/em\u003e and neurodevelopmental and neuropsychiatric disorders. We identified 25 individuals with a clinical phenotype characterized by a high prevalence of DD/ID, ASD traits, ADHD traits, other psychiatric disorders, and drug resistant epilepsy. These clinical observations are in line with the phenotype observed in the \u003cem\u003ecmip\u003c/em\u003e mutant zebrafish model, which demonstrated (temperature-dependent) alterations in locomotor activity in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e⁺/⁻\u003c/sup\u003e and \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e larvae and spontaneous epileptiform activity in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/⁻\u003c/sup\u003e larvae.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eClinical and genetic findings\u003c/h2\u003e \u003cp\u003eWe report 25 individuals with NDD linked to \u003cem\u003eCMIP\u003c/em\u003e, including six individuals with a SNV. Despite phenotypic heterogeneity, developmental delay (20/25, 80%), intellectual disability (18/25, 72%) and neuropsychiatric features (20/25, 80%) were highly prevalent. Surprisingly, no clear differences in clinical presentation were observed between individuals harbouring single gene mutations and those with large deletions in our cohort.\u003c/p\u003e \u003cp\u003eNeurological examination was abnormal in half of the cohort, characterised by movement disorders, including postural tremor, myoclonus and dyskinesia, but also nonspecific findings such as balance disturbances, clumsiness and mild hypotonia.\u003c/p\u003e \u003cp\u003eIntellectual functioning ranged from normal to severe ID, likely reflecting variable expressivity, genetic background, and environmental influences, as seen in other NDD-associated genes.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e The high prevalence of psychiatric comorbidities in \u003cem\u003eCMIP\u003c/em\u003e is remarkable, compared to other individuals with NDD, as illustrated by internalising or externalising symptoms in up to 61% of school-age children with NDD.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e When considering a specific genetic etiology, the high rate of psychiatric comorbidities observed in our cohort parallels findings in fragile X syndrome, where anxiety and anxiety disorders are reported in up to 86% of individuals and mood instability and aggression occur in up to 58% of individuals.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e The involvement of \u003cem\u003eCMIP\u003c/em\u003e in the pathophysiology of psychiatric disorders is further supported by previously reported associations of \u003cem\u003eCMIP\u003c/em\u003e polymorphisms and schizophrenia.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e It could be that other factors, both environmental as well as genetic (other than \u003cem\u003eCMIP\u003c/em\u003e), contribute to the high prevalence of psychiatric disorders in our cohort.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eEpilepsy, although not universal, was another prominent feature in nine individuals (36%). It is striking that in cases 2 and 3, in which epilepsy was most severe, a regression was reported in all domains, after the onset of their seizures, as often is seen in other DEE.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e The remaining individuals had absence seizures or BTCS, which were most likely genetic generalized epilepsies, supported by generalized epileptic abnormalities on EEG in some.\u003c/p\u003e \u003cp\u003eThe high prevalence of gastro-intestinal manifestations observed in this cohort aligns with prior findings in children with ASD, where the occurrence of gastro-intestinal disorders is over four-fold increased higher compared to individuals without ASD.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Because of the high frequency of failure to thrive in this cohort, clinicians should be aware of this potential comorbidity in \u003cem\u003eCMIP\u003c/em\u003e-related NDD and regular follow-up of weight and dietary habits is advised.\u003c/p\u003e \u003cp\u003eCollectively, the broad phenotypical spectrum of neurological and neuropsychiatric features of \u003cem\u003eCMIP\u003c/em\u003e is in parallel with other neurologic disorders linked to adaptor proteins, in which the clinical picture also includes abnormal gait, spasticity, hypotonia and muscle weakness.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eNo alternative pathogenic drivers beyond\u003c/b\u003e \u003cb\u003eCMIP\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmong individuals with CNVs spanning \u003cem\u003eCMIP\u003c/em\u003e, several additional protein coding genes were co-deleted. To evaluate their potential contribution, we systematically reviewed each gene (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). A total of ten disease-associated genes were identified \u003cem\u003e(BCO1, DNAAF1, GAN, GCSH, MAF, MBTPS1, MLYCD, PLCG2, SLC38A8, WWOX\u003c/em\u003e), of which only \u003cem\u003eBCO1, MAF, and PLCG2\u003c/em\u003e are autosomal dominant. The remainder are autosomal recessive disorders.\u003c/p\u003e \u003cp\u003eNearly all these genes lacked a known association with neurodevelopmental and neuropsychiatric phenotypes. Although \u003cem\u003eMAF\u003c/em\u003e is associated with intellectual disability in Ayme-Gripp syndrome, the absence of congenital cataracts and hearing loss in the present cohort makes \u003cem\u003eMAF\u003c/em\u003e involvement unlikely.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eGAN\u003c/em\u003e causes giant axonal neuropathy, which may include ID with disease progression, but none of the individuals in this cohort had a phenotype related to it.\u003csup\u003e41\u003c/sup\u003e \u003cem\u003eWWOX\u003c/em\u003e, is associated with developmental and epileptic encephalopathy and has been proposed to be a weak ASD risk factor in heterozygous individuals.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Since GAN and WWOX are autosomal recessive, the heterozygous deletions in our cohort are unlikely to be pathogenic.\u003c/p\u003e \u003cp\u003eTaken together in our cohort, we found no alternative pathogenic variants, and the few co-deleted genes identified showed only limited or indirect evidence of pathogenicity. This absence of other genetic drivers strengthens the genotype\u0026ndash;phenotype correlation, indicating that \u003cem\u003eCMIP\u003c/em\u003e disruption is the main driver of the neurodevelopmental and neuropsychiatric features irrespective of the size of the deletion.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInsights from the\u003c/b\u003e \u003cb\u003ecmip\u003c/b\u003e \u003cb\u003emutant zebrafish model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the functional consequences of \u003cem\u003eCMIP\u003c/em\u003e loss of function and to support the pathogenicity of human \u003cem\u003eCMIP\u003c/em\u003e variants, a zebrafish \u003cem\u003ecmip\u003c/em\u003e mutant model was characterized. Mutant larvae displayed altered locomotor activity and spontaneous epileptiform brain activity, paralleling patient observations. The accelerated habituation observed in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e and \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e may result from altered neural circuit function due to \u003cem\u003ecmip\u003c/em\u003e loss, a developmental delay in environmental adaptation, or underlying hypotonia as also observed in CMIP patients. The subsequent observed hypoactivity has already been reported in several genetic zebrafish epilepsy models.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Remarkably, the presence of spontaneous epileptiform events was only observed in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e, suggestive of heterozygous, rather than homozygous, loss of \u003cem\u003ecmip\u003c/em\u003e predisposing to neural hyperexcitability, which mirrors the clinical observations. Taken together, the zebrafish data provide functional evidence that \u003cem\u003ecmip\u003c/em\u003e disruption causes behavioral hypoactivity and increased seizure susceptibility, recapitulating key features of \u003cem\u003eCMIP\u003c/em\u003e-associated NDDs. Clinically, only a subset of \u003cem\u003eCMIP\u003c/em\u003e patients first presented with epilepsy. Although epilepsy is not the most prominent feature of a \u003cem\u003eCMIP\u003c/em\u003e-related NDD, clinicians should be aware that different seizure types may occur, that seizures can be difficult to treat and may be accompanied with developmental regression. Notably, \u003cem\u003ecmip\u003c/em\u003e expression was markedly reduced at the RNA and protein level in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e larvae, although the latter was not completely abolished. The \u003cem\u003ecmip\u003c/em\u003e gene is not annotated as duplicated in zebrafish, and no alternative isoforms have been documented to account for residual expression. However, stopcodon readthrough has been reported in zebrafish, which may explain the unexpectedly high protein levels in the \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e larvae.\u003csup\u003e43\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe role of\u003c/b\u003e \u003cb\u003eCMIP\u003c/b\u003e \u003cb\u003ein neurodevelopment and epilepsy\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe exact pathophysiological function of \u003cem\u003eCMIP\u003c/em\u003e in neurodevelopment has yet to be fully elucidated. However, its importance as an adaptor protein involved in neuronal migration, cytoskeletal remodelling and synaptic formation, particularly via interactions with binding partners such as filamin A and the PI3K/Akt/mTOR pathway, emphasizes the central role of \u003cem\u003eCMIP\u003c/em\u003e in neurodevelopmental processes.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Future research would benefit from focusing on which specific pathways are involved in the pathophysiology of \u003cem\u003eCMIP\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eClinical data were collected retrospectively, and complete information was not available for all individuals. Despite these missing data, we were able to present a robust overview of the clinical phenotype. It is important to acknowledge that the relatively limited cohort size precluded a detailed analysis of genotype-phenotype correlations. In the present study, we included only individuals with deletions or nonsense variants; however, missense variants have also been reported in DECIPHER, GeneMatcher and once in a large cohort of children with neurodevelopmental disorders.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e Some of these exhibit phenotypes similar to those observed in our cohort. Future research should therefore also address the potential pathogenicity of these variants, ideally including functional \u003cem\u003ein vitro\u003c/em\u003e studies.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our findings establish \u003cem\u003eCMIP\u003c/em\u003e as a strong candidate gene for NDD. Thus. we suggest considering \u003cem\u003eCMIP\u003c/em\u003e as a cause for DD-related phenotypes, associated with a wide range of psychiatric disorders (including ASD and ADHD), epilepsy, gastrointestinal features and potentially movement disorders. Including \u003cem\u003eCMIP\u003c/em\u003e in next generation sequencing-panels will lead to the discovery of additional individuals, allowing further insights in the phenotype, including \u003cem\u003eCMIP\u003c/em\u003e variant (re)classification. Follow-up studies will refine the clinical spectrum of this disease and the molecular pathways leading to the pathogenicity of \u003cem\u003eCMIP\u003c/em\u003e.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplemental material.\u003c/p\u003e \u003c/div\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests. AD received funding from the KU Leuven Fund for Childhood Epilepsy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.leuvenkinderepilepsiecentrum.be/en\u003c/span\u003e\u003cspan address=\"https://www.leuvenkinderepilepsiecentrum.be/en\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003ch2\u003eEthical approval\u003c/h2\u003e \u003cp\u003eAll data were collected and processed in accordance with the European General Data Protection Regulation (GDPR; EU 2016/679) and Belgian national legislation. Site-specific informed consent was obtained to all participants. The study was approved by the Institutional Review Board of Antwerp University Hospital (B300201316250).\u003c/p\u003e \u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eConceptualization: MDW, MVDL, AJ, FK; MDW collected clinical data; MVDL, AD, DC and FK designed and conducted the experiments; all co-authors contributed to the writing-review and editing of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe are grateful to all the individuals and their family who cooperated in this study, as well as to their referring physicians. Jolien Huyghebeart and Claudio d\u0026rsquo;Incal for helping with the primer designs and Western blotting experiments. We thank Dr. Nathalie Van der Aa, MD for bringing the first case with a \u003cem\u003eCMIP\u003c/em\u003e deletion to our attention.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOllero M, Sahali D. 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A Collaborative Psychiatric-Genetics Inpatient Care Delivery Model Improves Access to Clinical Genetic Evaluation, Testing, and Diagnosis for Patients With Neurodevelopmental Disorders. \u003cem\u003eFront Genet\u003c/em\u003e. 2022;13:901458. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fgene.2022.901458\u003c/span\u003e\u003cspan address=\"10.3389/fgene.2022.901458\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"autism spectrum disorder, psychiatric disorder, language delay, intellectual disability, epilepsy, zebrafish","lastPublishedDoi":"10.21203/rs.3.rs-8316803/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8316803/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eCMIP\u003c/em\u003e, a c-maf inducing protein that plays a key role in cytoskeletal remodeling, neuronal migration and synaptic formation, was first associated with specific language impairment and autism through the identification of a deletion in a single patient in 2012. Since then, only two additional individuals with \u003cem\u003eCMIP\u003c/em\u003e deletions have been reported, both sharing features of autism and gastrointestinal features. However, a firm causal relationship between variants in \u003cem\u003eCMIP\u003c/em\u003e and neurodevelopmental disorders has not yet been established. In this multicentre cohort study, we identified 25 individuals with \u003cem\u003eCMIP\u003c/em\u003e-related neurodevelopmental disorders, 22 of whom have not been previously reported. Of these, seven individuals carried heterozygous loss-of-function \u003cem\u003eCMIP\u003c/em\u003e single nucleotide variants, while the other 18 individuals had a complete or partial deletion of \u003cem\u003eCMIP\u003c/em\u003e, some involving adjacent genes. The clinical phenotype was variable with a high prevalence of developmental delay (20/25), autism spectrum disorder features (13/25), attention-deficit/hyperactivity disorder features (11/25) and other psychiatric disorders (15/25). Epilepsy was present in nine individuals (9/25), of whom three had therapy-resistant seizures. To study the pathogenicity of \u003cem\u003eCMIP\u003c/em\u003e variants, a \u003cem\u003ecmip\u003c/em\u003e mutant zebrafish model carrying a premature stop codon was investigated. These mutants showed temperature-dependent altered locomotor activity suggestive of seizure-like behavior, which was confirmed by spontaneous epileptiform discharges in \u003cem\u003ecmip\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e mutant zebrafish larvae. Our patient cohort and the zebrafish data establish \u003cem\u003eCMIP\u003c/em\u003e as a gene implicated in neurodevelopmental and neuropsychiatric disorders. We recommend inclusion of \u003cem\u003eCMIP\u003c/em\u003e in the genetic work-up of neurodevelopmental delay, with or without autism or psychiatric disorders and epilepsy.\u003c/p\u003e","manuscriptTitle":"CMIP as a novel candidate gene for neurodevelopmental and neuropsychiatric disorders","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 10:39:37","doi":"10.21203/rs.3.rs-8316803/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-03-16T10:48:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-04T13:55:26+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-17T17:04:14+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-09T15:35:12+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-04T07:58:09+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-01-12T18:22:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-05T11:52:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Human Genetics","date":"2025-12-23T09:07:57+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-12-11T10:10:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-09T11:02:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-human-genetics","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ejhg","sideBox":"Learn more about [European Journal of Human Genetics](http://www.nature.com/ejhg/)","snPcode":"41431","submissionUrl":"https://mts-ejhg.nature.com/cgi-bin/main.plex","title":"European Journal of Human Genetics","twitterHandle":"@ejhg_journal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4bf3925e-9053-4221-83e7-91287f706019","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61018716,"name":"Health sciences/Diseases/Neurological disorders/Neurodevelopmental disorders/Autism spectrum disorders"},{"id":61018717,"name":"Health sciences/Diseases/Psychiatric disorders"},{"id":61018718,"name":"Health sciences/Diseases/Neurological disorders/Epilepsy"}],"tags":[],"updatedAt":"2026-04-10T08:42:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 10:39:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8316803","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8316803","identity":"rs-8316803","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}