# NOVA1/2 genes and alternative splicing in neurodevelopment

Amélie Piton

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Amélie Piton. NOVA1/2 genes and alternative splicing in neurodevelopment. Current Opinion in Genetics and Development, 2025, 93, pp.102373. 10.1016/j.gde.2025.102373. hal-05494598

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Review

# NOVA1/2 genes and alternative splicing in neurodevelopment

Amélie Piton<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="4" />,<fnref n="5" />

<figure><img src="image_3.png" /></figure>

NOVA1 and NOVA2 are neuron-specific RNA-binding proteins essential for alternative splicing (AS), influencing neurodevelopment by regulating transcript diversity. These proteins recognize YCAY sequences on pre-mRNA, regulating exon inclusion or skipping, intron retention, and alternative polyadenylation. Despite their 75% sequence identity, NOVA1 and NOVA2 exhibit distinct spatiotemporal expression patterns and target specificities, with NOVA2 predominantly expressed in cortical regions and NOVA1 in the cerebellum and spinal cord. *De novo* truncating variants in *NOVA2* are responsible for a severe neurodevelopmental disorder (NDD), characterized by intellectual developmental disorder, motor delay, autistic features, and corpus callosum hypoplasia. Loss of *Nova2* in animal models results in brain development anomalies, such as corpus callosum agenesis in mice, which mirrors the human neurodevelopmental phenotype. If direct evidence remains limited, emerging data suggest that mutations in *NOVA1* might also be involved in neurological disorders. The contribution of other mRNA-binding proteins to NDD further underscores the critical role of regulation of RNA processing in neurodevelopment. This review explores the diverse functions of NOVA proteins, their impact on AS during brain development, and their implications in brain disorders.

## Addresses

Corresponding author: Piton, Amélie (piton@igbmc.fr)

**Current Opinion in Genetics & Development** 2025, 93:102373

This review comes from a themed issue on **Molecular and Genetic Basis of Disease**

Edited by Naiara Akizu and Joseph Gecz

For complete overview of the section, please refer to the article collection, "Molecular and Genetic Basis of Disease (2025)"

Available online 23 June 2025

https://doi.org/10.1016/j.gde.2025.102373

0959-437X/© 2025 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

Alternative splicing (AS), allowing the generation of multiple protein isoforms from single genes, is an essential mechanism ensuring transcriptomic diversity. AS regulation plays a fundamental role in organ development and differentiation [1,2]. In brain development, AS events are tightly regulated in a spatiotemporal manner [3,4]. The neuro-oncological ventral antigens 1 and 2 (NOVA1 and NOVA2) are RNA-binding proteins (RBPs) highly expressed in the brain, known to regulate AS events essential for brain development and function. These proteins share 75% sequence identity and contain three K-homology (KH) domains that recognize YCAY sequence on mRNA, located near exon-intron junctions [5-7]. Depending on the distribution of these motifs, NOVA proteins can promote either inclusion or exclusion of the target exon: binding in the exon or just upstream usually favors exon skipping by blocking U1 snRNP recruitment, while binding to a YCAY cluster localized downstream the target exon increases its inclusion by promoting spliceosome assembly [8]. In addition to exon regulation, NOVA proteins can also regulate intronic retention [9,10] and alternative polyadenylation [11] (Figure 1). NOVA2 has also been recently shown to regulate the generation of circular RNA, as their levels decrease in the embryonic cortex of *Nova2* knockout (KO) mice [12]. Although NOVA proteins are predominantly expressed in brain tissue, lower expression has been reported in non-neuronal tissues and cancer cells (Gtex [13]). This review focuses on their role in brain development and in their involvement in neurodevelopmental disorders (NDDs). NOVA proteins are highly expressed in different brain

<footnote n="1">¹Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France</footnote>

<footnote n="2">²Centre National de la Recherche Scientifique, UMR7104, Illkirch, France</footnote>

<footnote n="3">³Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France</footnote>

<footnote n="4">⁴Université de Strasbourg, 67 400 Illkirch, France</footnote>

<footnote n="5">⁵Genetic diagnosis laboratory, Strasbourg University Hospital, 67 090 Strasbourg, France</footnote>
<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>

Illustration of the different mechanisms of mRNA regulation by NOVA proteins in the brain. NOVA proteins are involved in the posttranscriptional regulation of mRNA at different levels: (a) inclusion or exclusion of alternative exons: for instance, in mouse brain and in human neural stem cells, loss of NOVA2 reduces the inclusion of exon 26 in the *Neo1* transcripts [17,19], (b) regulation of intron removal: some introns can be retained in the transcript, such as *Itpr1* intron 23 in mouse cortical excitatory neurons, when NOVA proteins are absent [9], (c) regulation of alternative polyadenylation sites, as observed in the *Slc8a1 3'UTR* where NOVA2 improves the use of a distal poly(A) site in mouse brain [11], (d) competition with miRNA binding to mRNA, illustrated by stabilization of *Impact* mRNA by NOVA1 in mouse hypothalamic neurons [16], (e) generation of circular RNA (circRNA), such as *circEfnb2*, through back-splicing mechanisms facilitated by NOVA proteins [12]. Figure Created with BioRender.com.

regions in rodents, with *Nova2* being more abundant in cortex, hypothalamus, and striatum and *Nova1* in ventral spinal cord [6,14]. In human, *NOVA1* is highly expressed in the cerebellum, especially at postnatal stages, while *NOVA2* is more expressed than *NOVA1* through the different cortical regions and the limbic system in adult (Figure 2) throughout all developmental stages (data from https://www.brainspan.org/).

## Loss of NOVA proteins causes brain alterations in animal models

Biallelic *Nova1* KO mice are born with a normal appearance but experience failure to thrive, motor

dysfunction, and premature death, accompanied by increased apoptosis of neuronal cells in the spinal cord and brainstem [15]. A conditional *Nova1* KO model in inhibitory neurons (*Gad2Cre-Nova1*) showed growth retardation, learning and memory deficits, and impaired anxiety, with surviving mice [16]. Biallelic *Nova2* KO mice exhibit growth retardation and premature death similar to *Nova1* mutants but also display agenesis of the corpus callosum [17]. *Nova2* loss impairs neuronal migration of late-born neurons in the cortex and calbindin-positive migrating neurons in the cerebellum [18]. Heterozygous *Nova2 +/-* mice, unlike homozygous KO, do not die or exhibit a severe phenotype. However, they
<figure><img src="image_5.png" /><figcaption>Figure 2</figcaption></figure>

Expression of NOVA proteins in human brain. Data from Brainspan (https://www.brainspan.org/) were used to represent the expression of *NOVA1* and *NOVA2* mRNA across the different human brain regions. Are also indicated the constraint metrics for both genes: intolerance of the genes to heterozygous loss of function (pLI), loss-of-function observed/expected upper bound fraction (LOEUF), and constraint on missense variants (Z-score *mis*) calculated from gnomAD database v4.1.0 (https://gnomad.broadinstitute.org/).

present frequent synchronous cortical discharges suggestive of epilepsy. *Nova2* KO mice also show motor innervation defects in the diaphragm and auditory ef-ferent innervation defects [17].

To further elucidate NOVA2's role in brain, different conditional models of *Nova2* loss (cKO) in specific brain regions have been studied [9]. For example, inactivation of *Nova2* in excitatory neurons (*Emx1Cre-Nova2*) leads to premature death, cortical laminar disorganization, and hippocampal structural alterations [9]. In cerebellar Purkinje cells (*Pcp2Cre-Nova2*), *Nova2* loss results in dendritic shrinkage, cell death, cerebellar atrophy, and motor deficits [9].

In zebrafish, morpholino-induced *Nova2* inactivation causes brain anomalies, including reduced axon numbers crossing between the two optic tecta and a global decrease in brain size, which is rescued by expressing wild-type NOVA2 mRNA [19].

**NOVA proteins regulate alternative splicing events essential for proper brain development and functioning**

Thanks notably to the works of Robert Darnell's team and the development of high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation method, hundreds of splicing events regulated by NOVA proteins have been identified in mice brain over the past decade, in the whole brain [20], in different brain subregions [11], in neuronal types [9], or at different stages of brain development [18]. NOVA-regulated splicing events of neurotransmitter receptors (e.g. glycine receptor alpha2, GABAA receptor γ2, Grik2, Grin1, dopamine D2 receptor, etc) [11,15,20-23], cation channels (*Cacna1a*, *Cacna1b*, *Scn5a*, etc) [11,20,24], scaffolding proteins (*Cask*, *Ank3*, *Gphn*, etc) [17,20], cytoskeleton-associated proteins (*Sept8*) [25], and proteins involved in cell-cell adhesion (*Aplp2*, *Epha5*, etc) [8,20]. Although NOVA1 and NOVA2 bind the same mRNA motifs,

experiments performed by Darnell's team showed that they can regulate distinct targets and specific biological processes. NOVA2 regulates more AS events than NOVA1 [9,17], particularly in genes encoding proteins involved in axonal guidance (e.g. *Dcc*, *Neo1*, *Roundabout Guidance Receptor 2* [Robo2], *Slit2*, etc [17]).

By using a combination of *Nova2* cKO models in different neuronal subtypes and a cTag-CLIP method allowing to identify mRNA targets of NOVA2 from different neuronal populations, Darnell's team showed that NOVA2 can regulate AS in different manner depending on the brain regions, the neuronal cell types and the stages of development [9,11,18]. In particular, NOVA2 modulates the splicing of *Dab1*, a key player in Reelin signaling, by inhibiting the inclusion of exons 7b and 7c [18]. The abnormal ratio between *Dab1.7bc* and *Dab1* caused by *Nova2* loss leads to alterations in neuronal migration during cortical development. This splicing regulation is specific to excitatory neurons and does not occur in inhibitory neurons [9]. NOVA2 also regulates intron removal in a cell type—specific manner: it regulates, for instance, the removal of intron 23 from the *Itrp1* gene, a gene involved in the spino-cerebellar ataxia SCA29, and this regulation occurs in cortical excitatory neurons but not in Purkinje neurons [9]. These differences in the regulation of the same transcripts across the neuronal cell types by NOVA2 might depend on the expression ratio of the other splicing regulatory factors and might contribute to generate neuronal-types-specific mRNA regulatory diversity.

The AS regulation of neurotransmitter receptors and in particular N-methyl-D-aspartate receptors such as *GRIN1* by NOVA proteins support a role in synaptic function and plasticity [20]. An alteration of the long-term potentiation (LTP) of the slow inhibitory postsynaptic current was observed in hippocampus of *Nova2* KO mice, with no effect on the excitatory postsynaptic current LTP [26]. Conversely, neuronal activity can regulate NOVA proteins, as shown in inhibitory
somatostatin interneurons where *in vivo* electro-
convulsive stimulation leads to an increase of both
NOVA protein expression and nuclear localization [27].
In the rat striatum, induction of seizures using an agonist
of muscarinic acetylcholine receptors leads to an increase
of *Nova1* expression together with a slight decrease of
*Nova2* expression [14].

Most studies on the role of NOVA proteins in brain
development have been conducted in mice. A transient
knockdown of *NOVA2* using small interfering RNA
performed in human neural stem cells (hNSC, cortical
neuron progenitors) was performed recently and re-
vealed changes in AS events of genes encoding trans-
membrane proteins, proteins of the extracellular matrix
and proteins involved in cytoskeleton organization and
axon projection [19]. We observed a strong conservation
in AS events regulation between mice and human, with
similar target genes (e.g. *DAB1*, *SLIT2*, *SORBS1*, etc)
but also target exons (*NEO1*).

NOVA2 regulates alternative splicing events
involved in control of axon guidance of
commissural neurons

Regulation of *Neo1* exon 26 inclusion, previously iden-
tified in the mouse brain, was confirmed in human cor-
tical progenitors [19]. This AS event, which regulates the
inclusion of 53 amino acids in the intracellular domain of
this transmembrane receptor, is highly conserved across
species and was initially described in chick embryo [28].
*Neo1* and its paralog *Dcc* encode transmembrane re-
ceptors involved in the Netrin-1-dependent regulation
of axonal outgrowth and pathfinding, important for the
establishment of commissures in the brain and spinal
cord [29–31]. In the mouse brain, NOVA proteins reg-
ulate also *Dcc* AS: the isoform containing a long version
of exon 17, encoding a 40-amino acid sequence (residues
820–839) located between the fibronectin type-III do-
mains 4 and 5 in the extracellular part of DCC, is re-
duced in the dorsal spinal cord of double KO *Nova1/
Nova2* mutants [32] and in the cortex of *Nova2* KO mice
[17] (Figure 3). We could not observe this effect in
hNSC, likely because this long isoform is barely ex-
pressed in these cells (data not shown from Mattioli
et al., 2020). Interestingly, *Dcc* Ko mice display com-
missural defects in the brain and the spinal cord
[31], while mutations in *Dcc* have been described in in-
dividuals with isolated agenesis of the corpus callosum
[33], with most mutations localized in the Netrin-1-
binding domain.

In addition to *Dcc* and *Neo1*, additional AS events af-
fecting proteins involved in the regulation of axonal
guidance have been observed after *Nova2* loss. For in-
stance, the *Robo2* gene shows increased inclusion of
exon 6b in the spinal cord of double KO mice [34] and in

the cortex of *Nova2* KO [17]. This AS event is critical for
proper timing of midline crossing [34]. AS regulation of
the Robo ligand *Slit2* was also observed with decreased
inclusion of exon28b in *Nova2* KO brain [17]. While a
slight, nonsignificant decrease in exon 28b inclusion was
observed in transcripts from hNSCs treated with si-
*NOVA2*, a significant decrease was detected in exon 9
inclusion, another known AS target in this gene [19].
Finally, decreased inclusion of exon 7 of Ephrin Re-
ceptor A5 (Epha5) was observed in *Nova2* KO mice [17],
whereas this gene is not expressed in hNSC. Disruptions
of these AS regulations, which are critical for axonal
projection, may explain the reduction in the number of
commissural axons are able to reach the midline ob-
served in animal models of *Nova2* loss [17,19].

NOVA proteins and neurological and
neurodevelopmental disorders

The first link between NOVA proteins and human dis-
ease came from the discovery of autoantibodies against
NOVA1 in patients with paraneoplastic opsoclonus-
myoclonus ataxia (POMA), a disorder causing motor
control loss [35,36]. Autoantibodies against NOVA2 were
later identified, possibly contributing to encephalopathy
and cortical deficits observed in some POMA patients
[6]. In 2020, Mattioli et al. reported six *de novo* frameshift
variants in the *NOVA2* gene in individuals with a severe
NDD, including intellectual developmental disorder,
motor delay, speech impairment, autistic features, hy-
potonia, feeding difficulties, spasticity or ataxic gait, and
corpus callosum thinning [19]. Before the identification
of *NOVA2* variants, most of the patients underwent a
genetic analysis of the UBE3A locus, suggesting that this
novel syndrome (now referenced in OMIM as #618859)
represents a differential diagnosis for Angelman syn-
drome. The variants clustered in the same GC-rich re-
gion at the beginning of the fourth and last exon of the
gene (Figure 4). They all led to the same novel reading
frame, resulting in a common C-terminal extension re-
placing the third RNA-binding domain (KH3). Func-
tional assays showed that variants reduce, but do not
abolish, binding to target mRNA sequences, affecting
AS regulation more mildly than a stop codon introduced
at the same position. This supports the hypothesis of a
hypomorphic effect of the mutations rather than a
complete loss-of-function that might be too deleterious.

Two years later, Scala et al. reported seven additional
pathogenic *NOVA2* variants in eight individuals with
NDD [37]. Four variants were located in the same re-
gion as those previously identified, leading to the same
C-terminal extension, helping to refine the clinical
spectrum associated to *NOVA2* pathogenic variants. This
confirmed, for example, that corpus callosum hypoplasia
is present in one-third of the patients, highlighting ax-
onal projection defects also seen in both mice and
<figure><img src="image_6.png" /><figcaption>Figure 3</figcaption></figure>

NOVA2 protein regulates alternative exons important for axonal guidance of commissural axons. (a) *Nova2* loss leads to impairments in axonal guidance, which can be visualized in zebrafish brain by a decrease in the number of axons crossing between the two optic tecta [19], in mice or in human by a corpus callosum agenesis [17] or hypoplasia respectively [19,37]. (b) Representation of the different AS regulates by NOVA2 in genes involved in regulation of axonal projection, Dcc, its close paralog Neo1, as well as ROBO2 receptor and its ligand Slit2. AS exon identified in mice by <sup>a</sup>Saito et al. [17], <sup>b</sup>Leggere et al. [32], <sup>c</sup>Mattioli et al. [19], and <sup>d</sup>Johnson et al. [34] in mice (Mm) or in human (Hs) and localization of the encoding amino acids in the corresponding protein (pink arrow). Figure created with **BioRender.com**.

zebrafish [17]. However, no other obvious cortical mal-
formations were reported in patients, despite what was
found in mice [18] and the fact that *DAB1* transcript is
regulated by NOVA2 in both mice and human cells.
This could be possibly due to the presence of a single
disrupted *NOVA2* allele in human patients. For the first
time, early truncating variants occurring in the first and
the second KH domain (position 86 and 175) were also

observed in two patients with NDD, challenging the
earlier hypothesis that a complete haploinsufficiency
would be too deleterious to be viable. Moreover, a *de
novo* distal truncating variant occurring in the KH3 do-
main was also identified. This variant showed impair-
ments in the regulation of AS events, but with a milder
effect than the previously reported variants, despite the
patient presents a severe form of NDD.
<figure><img src="image_7.png" /><figcaption>Figure 4</figcaption></figure>

De novo truncating variants identified in the *NOVA2* gene in individuals with NDDs. Schematic representation of the NOVA2 protein with its nuclear localization signal (amino acids 10-26) in green and its three KH-type RNA-binding domains KH1 (32-99), KH2 (130-196), and KH3 (406-473) in blue. The de novo truncating variants reported in patients with NDD are indicated: <sup>a</sup>variants reported in Mattioli et al. [19]; <sup>b</sup>variants reported in Scala et al. [37]; <sup>c</sup>variants reported as pathogenic in ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/). The variants resulting in the same C-terminal extension of more than hundred amino acids are indicated in red.

The partially overlapping functions of NOVA proteins raise the question of a potential compensatory mechanism, where NOVA2 loss might be compensated by an increase in NOVA1 expression. Saito et al. showed that NOVA1 protein is slightly increased in *Nova2* KO mice, but the mice still exhibit brain anomalies and growth delays, suggesting that this increase cannot fully compensate for the loss of NOVA2 [17]. In humans, the restricted access to brain tissue prevents the investigation of any potential compensatory effect in patients, but no increase of *NOVA1* was observed following *NOVA2* knockdown in human neural stem cells *in vitro*, at least at the mRNA level [19].

To date, *de novo* variants in *NOVA1* have not yet been reported as causing NDD, but given its loss-of-function intolerance (Figure 2), which is even stronger than *NOVA2*, truncating variants could potentially be disease causing. It is important to note that frameshift variants, which would be located in the same region as the *NOVA2* variants, would result, in the case of *NOVA1*, in an early stop codon rather than a long C-terminal extension. Recently, a 2 Mb deletion encompassing *NOVA1* as the only coding-protein gene was identified in an individual with developmental delay, hypotonia, and behavioral problems [16]. However, this is a unique observation, and there is no detailed information about the variant inheritance or the presence of any additional genetic variants in this individual. The publication of additional cases would therefore be necessary to conclude whether *NOVA1* haploinsufficiency is responsible for an NDD. The pattern of NOVA1 expression throughout life would more strongly support an implication in adult neurological diseases rather than in neurodevelopment diseases.

*NOVA1* has been linked to neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). In induced pluripotent stem cell-derived motor neurons obtained from ALS patient, an increase level of *NOVA1* (and to a lesser extend *NOVA2*) is observed in the insoluble fraction before the accumulation of TDP-43 in the cytoplasm, followed by a global decrease afterward, confirmed in mice models [38]. This suggests that NOVA proteins might contribute to the mRNA dysregulations observed in ALS patients.

## Implication of additional alternative splicing RNA-binding proteins in neurodevelopmental disorders

Additional RBPs involved in the regulation of AS have also been implicated in NDDs, such as *RBFOX1* or *CELF2*. The *RBFOX1* gene, also known as *A2BP1*, was described as a candidate gene for NDD since the identification of three patients harboring a chromosomal translocation interrupting its coding sequence [39,40]. A downregulation of RBFOX1/A2BP1, as well as a dysregulation of the splicing of its target exons, was observed in brain of individuals with autism [41]. Recently, *de novo* missense variants in the RNA recognition motif of *RBFOX1* were reported in six individuals with variable degree of developmental delay [42]. Heterozygote variants in the *CELF2* (CUG-BP, Elav-like family) gene, including truncating variants and missense variants affecting the nuclear localization of the protein, were reported in five individuals with developmental and epileptic encephalopathy [43].

The other members of RBFOX (*RBFOX2*, *RBFOX3*) and CELF (*CELF1*) families have not been reported yet as
involved in human diseases while they are highly intol-
erant to loss-of-function but present a similar expression
profile compared to *NOVA1*, with an expression higher in
cerebellum than in cortex (source from ProteinAtlas,
https://www.proteinatlas.org/humanproteome/brain).

More generally, more than fifty 50 of 400 proteins in-
volved in splicing regulation [44] are encoded by genes
leading to NDD when mutated (e.g. *PUF60*, *PQBP1*,
*EFTUD2*, etc https://sysndd.dbmr.unibe.ch/). In addi-
tion, mutations in several small noncoding RNA essen-
tial components of the spliceosome, such as the recently
reported RNU4-2, are also mutated in NDD [45,46].

Conclusions

NOVA1 and NOVA2 proteins, as master regulators of AS, are critical for brain development and function throughout life. Their regulation of splicing events ensures proper neuronal differentiation and synaptic connectivity. Animal models demonstrate that loss of NOVA proteins causes developmental anomalies, while mutations in *NOVA2* are linked to a severe form of NDD in humans. Emerging evidence also implicates *NOVA1* in brain disorders, although further investigations are needed. Future research, such as using brain organoids, may clarify the precise consequences of NOVA protein dysfunction on brain development and function and help uncover potential treatments for splicing-related NDDs.

Data Availability

Review.

**Declaration of Competing Interest**

The authors declare that they have no known competing
financial interests.

**References and recommended reading**

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest

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37. Scala M, Drouot N, MacLennan SC, Wessels MW, Krygier M,

• Pavinato L, Telegrafi A, de Man SA, van Slegtenhorst M, Iacomino M, et al.: De novo truncating NOVA2 variants affect alternative splicing and lead to heterogeneous neurodevelopmental phenotypes. *Hum Mutat* 2022, **43**:1299-1313.

This study reported, 2 years after the initial publication, additional patients with developmental delay carrying *de novo* truncating variants in *NOVA2*, allowing to refine the molecular and clinical spectrum of this novel genetic NDD.

38. Krach F, Wheeler EC, Regensburger M, Boerstler T, Wend H, Vu AQ, Wang R, Reischl S, Boldt K, Batra R, et al.: Aberrant NOVA1 function disrupts alternative splicing in early stages of amyotrophic lateral sclerosis. *Acta Neuropathol* 2022, **144**:413-435.

The authors focused on identifying molecular defects occurring in the early stages of ALS in motoneurons derived from patients. Notably, they reported an increase in NOVA proteins within the insoluble protein fraction before TDP-43 cytoplasmic accumulation and identified AS changes enriched for NOVA-binding sequences. This highlights that NOVA-dependent AS dysfunctions may contribute to ALS pathogenesis, preceding the neuropathological TDP-43 hallmark of the disease.

39. Bhalla K, Phillips HA, Crawford J, McKenzie OLD, Mulley JC, Eyre H, Gardner AE, Kremmidiotis G, Callen DF: The de novo chromosome 16 translocations of two patients with abnormal phenotypes (mental retardation and epilepsy) disrupt the A2BP1 gene. *J Hum Genet* 2004, **49**:308-311.

40. Martin CL, Duvall JA, Ilkin Y, Simon JS, Arreaza MG, Wilkes K, Alvarez-Retuerto A, Whichello A, Powell CM, Rao K, et al.: Cytogenetic and molecular characterization of A2BP1/FOX1 as a candidate gene for autism. *Am J Med Genet B Neuropsychiatr Genet* 2007, **144B**:869-876.

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42. Li D, Wang Q, Bayat A, Battig MR, Zhou Y, Bosch DG, van Haaften G, Granger L, Petersen AK, Pérez-Jurado LA, et al.: Spliceosome malfunction causes neurodevelopmental disorders with overlapping features. *J Clin Invest* 2024, **134**:e171235.

The authors have compiled evidences for the implication of three genes encoding proteins involved in splicing (U2AF2, PRP19) and AS (RBFOX1) in NDDs. The number of patients described (around 40 for U2AF2) and the functional studies performed (neuritogenesis impairment in human neurons and drosophila models) allowed a robust molecular and clinical characterization of these novel genetic forms of NDD.

43. Itai T, Hamanaka K, Sasaki K, Wagner M, Kotzaeridou U, Brösse I, Ries M, Kobayashi Y, Tohyama J, Kato M, et al.: De novo variants in CELF2 that disrupt the nuclear localization signal cause developmental and epileptic encephalopathy. *Hum Mutat* 2021, **42**:66-76.

44. Seiler M, Peng S, Agrawal AA, Palacino J, Teng T, Zhu P, Smith PG, Cancer Genome Atlas Research Network, Buonamici S, Yu L: Somatic mutational landscape of splicing factor genes and their functional consequences across 33 cancer types. *Cell Rep* 2018, **23**:282-296 e4.

45. Merico D, Roifman M, Braunschweig U, Yuen RKC, Alexandrova R, Bates A, Reid B, Nalpathamkalam T, Wang Z, Thiruvahindrapuram B, et al.: Compound heterozygous mutations in the noncoding RNU4ATAC cause Roifman Syndrome by disrupting minor intron splicing. *Nat Commun* 2015, **6**:8718.

46. Chen Y, Dawes R, Kim HC, Ljungdahl A, Stenton SL, Walker S,

• Lord J, Lemire G, Martin-Geary AC, Ganesh VS, et al.: De novo variants in the RNU4-2 snRNA cause a frequent neurodevelopmental syndrome. *Nature* 2024, **632**:832-840.

This study reported variants in a small noncoding U4 small nuclear RNA, RNU4-2, as one of the most frequent cause of NDDs. It is the demonstration that frequent monogenic forms of NDD remain to be identified. Moreover, it illustrates the power of using variant recurrence (n.64_65insT is highly recurrent) and *de novo* inheritance to conclude that variants occurring in noncoding regions are disease causing. Finally, it confirms the implication of splicing dysregulations in NDD.