Neurodevelopmental disorders with language disorders and behavioral abnormalities (NEDLIB, OMIM#618917) are caused by heterozygous mutations in the glutamate ionotropic receptor AMPA type subunit 2 (GRIA2) gene on chromosome 4q32. The disease can lead to impaired mental development or developmental delay, poor or lack of language ability, abnormal gait, uncoordinated movement, and abnormal behaviors with autism characteristics such as stereotypes, compulsions, and repetitions. Some patients also show clinical features such as seizures and brain atrophy. The disease is onset in infancy, the clinical manifestations are highly heterogeneous, and most of them are de novo mutations (Salpietro et al., 2019) (Supplementary Table S1).

The glutamate receptor sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) is a ligand-activated ion channel that mediates the fast component of excitatory postsynaptic currents in neurons of the central nervous system (Krupp and Feltz, 1995; Kohda et al., 2000). The ion channel is composed of four related subunits: GLURA (GRIA1, OMIM*138248), GLURB (GRIA2, OMIM *138247), GLURC (GRIA3, OMIM*305915), and GLURD (GRIA4, OMIM*138246) (Gécz et al., 1999; Armstrong and Gouaux, 2000; Beyer et al., 2008; Allen et al., 2012). The GLURB subunit makes the channel almost impermeable to calcium ions (Ca2+) (Schoepfer et al., 1994; Utz and Verdoorn, 1997).

The patient and her parents went to the Medical Genetics Center of Gansu Provincial Maternity and Child Health Hospital for genetic testing. Informed consent was given according to the agreement approved by the Institutional Review Committee. The proband is a 3-year-old female whose parents were healthy and unrelated. The patient had difficulty feeding during the newborn period. Brain MRI suggested brain atrophy in the proband. She could walk independently but was slow in movement. The patient had stereotyped behavior (repeated practice of speaking) and compulsive behavior (bringing her own quilt). She did not speak continuous sentences and did not communicate with others. Her concentration was poor. She was stunted. The child had the ability to paint.

In this study, a heterozygous missense mutation in the GRIA2 gene c.1934T > G (p.Leu645Arg) was detected in the proband. The mutation was de novo, and the parents were wild-type (Figure 1; Supplementary Table S1). According to ACMG guidelines, this variant was rated as likely to be pathogenic (PS2+PM1+PM2+PP3). The GRIA2 gene (NM_001083619.1) contains 16 exons; part of exon 1 and exons 2–15 are involved in gene coding (Figure 2A). GRIA2 is a transmembrane protein with three main domains: ANF_recepter (green), Lig_chan-Glu_bd (red), and Lig_chan (blue). The three transmembrane (orange diamond) areas are all on Lig_chan. So far, most of the reported missense mutations have been concentrated in the Lig_chan domain. The mutation site Leu645Arg in this study was also in this region (Figure 2B). As shown in the transmembrane protein pattern diagram, the Lig_chan (blue) domain of GRIA2 protein (NP_001077088.2) is transmembrane three times. The Leu645Arg site (arrow) in this case is located in the second transmembrane region. Both the ANF_recepter (green) and Lig_chan-Glu_bd (red) domains are located in the extracellular region (Figure 2C). In the GRIA2 protein sequence alignment of 7 species (human, Norway rat, chimpanzee, house mouse, cattle, chicken, and tropical clawed frog), position 645Leu is located in the conserved sequence (Figure 2D). In the 3D structure of wild-type GRIA2 protein, 645Leu and 641Leu form a hydrogen bond (Figures 3A,B). In the 3D structure of the mutant GRIA2 protein, Leu at position 645 became Arg (645Arg), which formed two hydrogen bonds with 641Leu (Figures 3C,D).

We report a female patient with neurodevelopmental disorders with language impairment and behavioral abnormalities (NEDLIB), presenting with delayed language development and stereotyped and compulsive behaviors. This is caused by a de novo missense mutation in exon 12 of the GRIA2 gene, which has not been reported previously.

Ionic glutamate receptors (iGluRs) are ligand-gated ion channels activated by the glutamate neurotransmitter (Palmada and Centelles, 1998). iGluRs mediate most of the excitatory synaptic transmission in the central nervous system and play a key role in synaptic plasticity, which is especially important for learning and memory (Murphy-Royal et al., 2017). Gene mutations in iGluR subunits can cause developmental delay (DD), intellectual disability (ID), and autism spectrum disorders (ASDs) and other related neurodevelopmental disorders (NDDs) (Olde Loohuis et al., 2015; Moretto et al., 2018; Edfawy et al., 2019; Wilding and Huettner, 2020). According to ligand binding characteristics and sequence similarity, iGluRs are divided into 4 subtypes: AMPA receptor, alginate receptor, NMDA receptor, and delta receptor. α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are assembled from the four subunits of Glu1-4, and the most common form in the forebrain is Glu1/Glu2 heterotetramer (Herguedas et al., 2019).

The GluA2 subunit encoded by GRIA2 plays a major role in the regulation of AMPAR Ca2+ penetration and voltage rectification. GluA2 has three main domains. The receptor family ligand binding region (ANF_receptor) includes the extracellular ligand binding domain of many receptors. This family also includes bacterial amino acid binding proteins with known structures (Kuryatov et al., 1994). The ligated ion channel L-glutamate- and glycine-binding site (Lig_chan-Glu_bd) is sometimes called the S1 domain. It is the luminal domain upstream of M1, the first transmembrane region of a transmembrane ion channel protein. This region binds l-glutamate and glycine (Ishii et al., 1993; Yamakura and Shimoji, 1999). Another time-transmembrane domain, the ligand-gated ion channel (Lig_chan) includes four transmembrane regions of ionotropic glutamate receptors and NMDA receptors (Tong et al., 1995).

Recent studies have shown that new heterozygous mutations in the GRIA2 gene were found in 28 nonrelated neurodevelopmental abnormalities with language disorders and behavioral abnormalities, including 15 missense variants, 2 splice site variants, 1 nonsense variant, 1 in-frame deletion, and 2 frameshift mutations. These mutations occur in the entire gene. In this case, we discovered a de novo missense variant in the GRIA2 gene, which can lead to NEDLIB (OMIM: #138247). It is also reported that the nonsense mutation of p. Glu508Ter in the GRIA2 gene leads to the occurrence of childhood onset schizophrenia (COS) (Alkelai et al., 2021) (Supplementary Table S1). So far, 16 missense variants of the GRIA2 gene have been reported in 20 patients, including the p. Leu645Arg discovered this time. The phenotypes of patients with missense variants of the GRIA2 gene are shown in Table 1. All patients have developmental delay and language impairment. A considerable number of patients have ASD and abnormal behaviors. Some patients have seizures. Conservation analysis of the amino acid sequence shows that p. Leu645Arg is in a conservative sequence. Including the p.Leu645Arg variant, 12 missense variants are concentrated in the Lig_chan domain. This domain is the transmembrane region of the GRIA2 gene-encoding protein, which forms three transmembranes, and the p.Leu645Arg site is located in the second transmembrane region. It could be seen that the Lig_chan domain may be the hotspot mutation region of the GRIA2 gene.

Taken together, our research has expanded the GRIA2 gene mutation spectrum, indicating that the detection and diagnosis of NEDLIB through WES is feasible and effective.