The chromodomain helicase DNA-binding protein 2 (CHD2) is a member of the ATP-dependent chromatin remodelling family of proteins that are critical for the assembly and regulation of chromatin (Lamar and Carvill, 2018). The CHD2 gene is located on the long arm of chromosome 15 and encodes a member of the CHD family of proteins (Wilson et al., 2021). The CHD2 gene contains 42 exons that encode 1828 amino acids. The CHD2 protein is composed of several functional domains, including two chromodomains at the N-terminus, an ATPase/helicase domain, and a DNA-binding domain. The N-terminal region of CHD2, which contains tandem chromodomains, serves an auto inhibitory role in both the DNA-binding and ATPase activities of CHD2 (Liu et al., 2015).

In 2009, a microdeletion of 15q26.1 was identified in a 30-month-old female who had refractory myoclonic epilepsy with intellectual disability, growth retardation, and a distinctive facial appearance (Veredice et al., 2009). In 2013, the gene primarily responsible for the epileptic component, and the disruption of neural developmental directly caused by seizures that resulted from the 15q26 defect, was CHD2 (Suls et al., 2013; Capelli et al., 2012; Epi4K Consortium et al., 2013; Poot et al., 2013). Since then, several mutations in the CHD2 gene have been identified and associated with developmental delay, microcephaly, autism, and epilepsy (Epi4K Consortium et al., 2013; Poot et al., 2013; Carvill et al., 2013; O'Roak et al., 2014; Deciphering Developmental Disorders Study, 2017). Seizures typically develop as early as 6 months and before 4 years of age; the multiple seizure types include myoclonus, myoclonic-absence seizures, and drop attacks (Capelli et al., 2012; Lamar and Carvill, 2018; Nieto-Estevez and Hsieh, 2018; Chen et al., 2020).

This study reports the cases of eight patients with CHD2 gene mutations and describes their clinical characteristics, laboratory examinations, electroencephalogram (EEG) results, mutational genotypes, and mRNA expression levels of CHD2 and repressor element 1-silencing transcription factor (REST). To our knowledge, all CHD2 mutations reported so far are heterozygous and the vast majority are new mutations. At present, the mechanism of CHD2 mutation to autosomal dominance is not clear. In this paper, we will preliminarily study and speculate the cause. Besides we found that the expression of REST gene was significantly increased in patients with CHD2 mutation, but the specific mechanism is still unclear and needs further study.

Mutations in the CHD2 gene can lead to neurodevelopmental delay, intellectual disability, epilepsy, and behavioural problems. Hanly et al. (Hanly et al., 2021) analysed 56 CHD2 gene mutations through a literature search and found that 80.3% (45 cases) of the patients had epilepsy; the incidence of epilepsy in our study was 87.5%. While most CHD2 mutations are de novo, a few are inherited from asymptomatic parents. To our knowledge, only Petersen et al. (Petersen et al., 2018) has reported a case of an inherited symptomatic CHD2 mutation, which affected both mother and daughter.

A search of the Human Gene Mutation Database (2020.6) shows that 128 cases of CHD2 mutation have been reported with 103 mutation sites. Missense mutations are the most common, followed by frameshift and nonsense mutations; splice mutations are uncommon, and other mutations are rare (Table 2). CHD2 is a relatively conserved gene, and many loci are relatively conserved in multiple species. These missense mutations that have been reported are mostly at these conserved loci. Among the 128 cases reported, the associated CHD2 mutations included c.693-1G>T (two cases), c.1809+1delG (three cases), c.1942C>T (two cases), c.2567A>G (three cases), c.2895_2898delAGAA (three cases), c.3521G>A (two cases), c.3734delA (two cases), c.3787dupG (two cases), c.4173dupA (four cases), c.4233_4236delAGAA (two cases), c.4799_4814delACCTTCACCCTCAGAA (three cases), c.4909C>T (four cases), c.4949dupG (three cases), c.5035C>T (three cases), and other mutations (one case). There seems to be no difference in the number of mutation sites of CHD2-related epilepsy in different domains (Figure 1).

Photosensitivity is often present, and self-induced seizures are seen in some patients (Suls et al., 2013; Galizia et al., 2015; Thomas et al., 2015). In this study, Case 1 and Case 2 showed photosensitivity, which is consistent with previous reports. Zebrafish larvae with a CHD2 knockout exhibit epileptic behaviour (Suls et al., 2013); CHD2 morpholino-injected larvae display body curvature, excessive body pigmentation, and a developmental delay (Suls et al., 2013). CHD2 ^+/− mice showed an increase in pyramidal neuron action potential discharge, a decrease in spinous process adaptation, significantly increased amplitude of micro-excitatory postsynaptic currents, and a significantly decreased frequency of micro-inhibitory postsynaptic currents. EEG recordings from the somatosensory neocortex of CHD2 ^+/− mice show increased frequency ranges of α, δ, θ, and γ waves, suggesting that CHD2 affects synaptic transmission of glutamate and γ-aminobutyric acid (GABA) and cortical rhythmgenesis in the adult hippocampus (Kim et al., 2018; Nieto-Estevez and Hsieh, 2018). This suggests that CHD2 mutation leads to a prominent reduction of GABA and an increase in neuronal excitability that results in seizures; this could be verified by further studies.

Two cases of the previously reported c5035C>T mutation were found in identical twin sisters (Cases 1 and 2). Their age of epilepsy onset was 4 years, and their clinical manifestations included complex partial seizures that initially presented as a persistent epileptic state and an EEG that suggested photosensitivity; these are consistent with previous reports (Wang et al., 2017).

Two cases of the p. Q 1392T*17 mutation were reported in this study, and five cases have been previously reported at this site (Lund et al., 2014; Galizia et al., 2015; Pinto et al., 2016; Chen et al., 2020), all of which were de novo and presented mainly as Lennox–Gastaut syndrome and autistic disorder. The reported manifestations included eyelid myoclonus, myoclonus, atonia, and atypical absence; all of the patients carrying this mutation had intellectual disability. However, the two cases we report in the present study had different clinical manifestations. One presented infantile spasm, while the other presented only mild intellectual disability and behavioural abnormalities, an abnormal EEG, and no seizures; these indicate that there are different clinical manifestations of the same gene mutation at the same site. Children with epilepsy inherited from asymptomatic fathers have been reported in the past (Chen et al., 2020). A CHD2 mutation that affected both mother and daughter has been reported with varying clinical manifestations (Petersen et al., 2018). This further supports the lack of a genotype–phenotype correlation among individuals with the same genetic variant, and suggests that CHD2 gene effects might be mediated by other mechanisms. It has been demonstrated that CHD2 mRNA is regulated in some tissues by the ubiquitous splicing regulator Rbfox2 (Cho et al., 2019), and CHD2 probably binds directly to REST genomic DNA to regulate the expression of REST (Shen et al., 2015). This suggests that different clinical manifestations may be related to these regulatory factors.

Mice with homozygous deletions of the C-terminus of CHD2 exhibit perinatal lethality, and heterozygous mice exhibit decreased survival rates, suggesting that CHD2 is an essential gene for normal development (Marfella et al., 2006). All the patients in this study had heterozygous mutations, and there were no homozygous or complex heterozygous mutations reported in the past, suggesting that the CHD2 gene is essential for human growth and development. Suls et al. (Suls et al., 2013) performed qPCR on two cases of nonsense mutations and found that the expression level was not decreased, which is consistent with the present study and suggests that the aberrant allele is not degraded by nonsense-mediated mRNA decay. Analysis using qPCR performed on a patient with a splicing mutation showed that both alleles were expressed (Suls et al., 2013). It is suggested that the CHD2 mutation does not affect the RNA expression level, and that the protein translated from the mutated CHD2 gene has some functionality; this mutant protein might affect the excitability of GABAergic neurons or other electrophysiological channels, resulting in increased neuronal excitability and leading to seizures.

Recent studies have shown that CHD2 is expressed throughout brain tissue, but varies in different cell subtypes at different developmental stages (Shen et al., 2015; Wilson et al., 2021). In mature mice (30 days after birth), CHD2 is significantly expressed in the olfactory bulb, neocortex, hippocampus, and cerebellum (Kim et al., 2018). In CHD2 ^+/− mice, there are fewer GABAergic interneurons in the somatosensory cortex, and the density of GABA neurons in the cortex is decreased; the loss of GABA intermediate neurons affects hippocampal memory (Kim et al., 2018). CHD2 is critical for embryonic and adult neuronal differentiation, synaptic properties, and memory; this partially explains the symptoms of CHD2 patients (Kim et al., 2018). Brain MRI of Case 8 in this study suggested that a reduction of the hippocampal volume might be related to the CHD2 mutation. During embryonic development, there is a decrease in proliferative cells in the ventricular zone and subventricular zone as well as in NKX2.1+ progenitor cells in the medial and caudal ganglionic eminence (Kim et al., 2018). CHD2 is highly expressed in the Pax6+ radial glia but rarely expressed in Tbr2+ intermediate progenitors, suggesting that CHD2 deficiency suppresses the self-renewal capacity of the radial glia and instead promotes premature neuronal differentiation (Shen et al., 2015; Lamar and Carvill, 2018). This early differentiation probably leads to rapid depletion of the progenitor pool, resulting in a smaller cortex and defects in later-born neurons (Homem et al., 2015; Lamar and Carvill, 2018). In human cortical interneurons derived from human embryonic stem cell cultures, Meganathan and others (Shen et al., 2015) found that CHD2 expression increases during cortical interneuron differentiation.

Knockout of the CHD2 gene in human embryonic stem cell–derived interneurons results in a decrease in the number of neurons and a reduction in the neurite length (Meganathan et al., 2017). Among the eight patients in this report, head MRI indicated widening of the cerebral sulcus in four cases; in one case head MRI imaging data could not be obtained; cranial MRI did not show abnormalities in the other three cases. Except for two patients that had normal development before the onset of epilepsy, the rest of the patients had different degrees of slowed mental development; the two patients experienced mental regression after the seizure onset.

CHD2 probably binds directly to the REST genomic DNA to regulate the expression of REST; the in vitro overexpression of REST rescues the defect in neurogenesis caused by CHD2 knockdown (Shen et al., 2015). To evaluate the relationship between CHD2 and REST in vivo, we measured the expression levels of REST mRNA in patients with CHD2 mutations. We found that the expression level of REST mRNA in the peripheral blood of patients with CHD2 mutations was significantly higher than that of the control (wild type) group. This suggests that the mutation of the CHD2 gene could result in high expression of REST, which might alleviate the clinical symptoms caused by the CHD2 mutation and cause the lack of a genotype–phenotype correlation mentioned above.

A total of six CHD2 mutation sites were reported among eight cases; of these, four mutation sites were previously unreported; this identifies new sites for pathogenic CHD2 mutations. The c.4173dupA mutation was reported in five previous cases, and two more cases are reported in this paper, suggesting that this site may be a hotspot for mutation. The pathogenesis of epilepsy caused by CHD2 is still unclear and may be related to decreased GABA activity. In this study, seven patients with epilepsy were treated with VPA to control their seizures, and it has been reported that VPA has good effect in the treatment of CHD2 mutation (Chen et al., 2020), but for patients with refractory epilepsy, a combination of medications or other methods are needed to control epilepsy. At present, the pathogenesis caused by CHD2 gene mutations is not clear, and further research is needed to elucidate the mechanism. Two possible causes of the phenotypic effects of a CHD2 gene mutation are as follows: 1) the mutant protein might still have some function and can lead to the dysfunction of another normal protein, and 2) the gene mutation results in reduced GABAergic channel activity, which can lead to seizures. These speculations need to be tested by further study.