1 Introduction

ANK2(HGNC:493) has 61 exons, is located on chromosome 4q25-q26, and encodes ankyrin B (ANKB), which is important for ion channel localization and membrane stabilization (Teunissen et al. 2023). There are two subtypes of ANKB: one protein is a wild-type 220 kDa protein that is expressed in multiple tissues, and the other protein is 440 kDa, also known as giant ANKB (gANKB), which is caused by alternative splicing. Mutations in this gene can cause various neurological disorders, such as autism spectrum disorders (ASDs) and epilepsy (EP) (Guissart et al. 2023).

ASD is a neurodevelopmental disease that usually manifests symptoms before the age of 3 years and is primarily characterized by impaired social communication, restricted interests, and stereotyped behaviors (Kawano et al. 2022; Hyman et al. 2020). The heterogeneity of the clinical manifestations of ASDs is caused by interactions among multiple genes, gene combinations, epigenetic factors, and environmental regulators (Oztenekecioglu et al. 2021). ANK2 mutations were identified as a functional neurospecific etiology of ASD in 2012 (Iossifov et al. 2012). However, reported cases of ASD associated with ANK2 mutations are rare. In this study, for the first time, we identified a novel ANK2 mutation in a patient with ASD and comorbid EP in China. We searched the literature for reports on ANK2 gene variations and summarized the clinical and genetic characteristics of these patients, aiming to improve the awareness of the gene among clinicians.

2 Materials and Methods

2.3 Past History and Family History: No Remarkable History

2.3.1 Physical Examination

His vital signs were stable, and he demonstrated normal development and good nutritional status. There was evidence of hyperactivity, restlessness, and frequent yelling. He exhibited intellectual impairment and was unable to communicate with others in a typical manner. Disconjugate gaze was observed, and the patient could not understand the instructions. The fundus, cardiopulmonary system, and abdominal examinations revealed no abnormalities. He exhibited normal muscle strength and muscle tone in the limbs, physiological reflexes were present, and pathological reflexes were not elicited.

2.3.2 Auxiliary Examination

The results of routine blood tests, routine urine tests, routine stool tests, and blood biochemical indices showed no obvious abnormalities. Video electroencephalography (Figure 1) revealed a poor background rhythm, increased fast waves, and multiple focal slow waves in each sleep period, particularly in the right frontal region. Cranial magnetic resonance imaging (MRI) revealed no abnormalities. ECG results were normal, and no abnormalities were detected via 24-h Holter ECG monitoring. The patient's score on the Pediatric Autism Rating Scale (2003) (Childhood Autism Rating Scale [CARS]) was 60, indicating a clinical diagnosis of severe ASD. Using trio whole-exome sequencing (WES), we identified a novel de novo mutation in the ANK2 gene: c.3007C>T, p.R1003* (NM_001148.6). To validate the authenticity of this mutation, Sanger sequencing was performed (Figure 2). According to the standards and guidelines of the American College of Medical Genetics and Genomics (ACMG), this variant was classified as pathogenic (PVS1 + PS2_Supporting+PM2_Supporting). Additionally, trioCNVseq, capillary electrophoresis testing for dynamic mutations in the fragile X syndrome gene FMR1, and mitochondrial genome analysis were performed, and no abnormal variations were detected.

3 Discussion

The ankyrin protein family includes ankyrin-R (AnkR), ankyrin-B (AnkB), and ankyrin-G (AnkG) (Sucharski et al. 2020; Bennett and Stenbuck 1979), which are encoded by ANK1, ANK2, and ANK3, respectively (Otto et al. 1991; Lambert et al. 1990). AnkR is important for maintaining the structural integrity and organization of red blood cell (RBC) membranes. Loss-of-function variants in ANK1 are associated with approximately 50% of hereditary spherocytosis cases and are characterized by abnormal shaping and fragility of RBCs (Narla and Mohandas 2017). ANK2 is highly conserved and can be expressed in multiple organs, especially in the brain, heart, and kidney. ANK2 has two major transcripts that produce 220-kDa (normal) and 440-kDa (giant) proteins. The 220-kDa AnkB protein has two isoforms, AnkB-188 and AnkB-212, both of which exhibit similar and unique patterns of alternative splicing: exons 7, 24, 30, and 40 are removed in both isoforms; exons 28, 38, and 46–50 are removed in AnkB-18; and exons 12–13 and 17 are removed in AnkB-212 (Sucharski et al. 2020; Wu et al. 2015). The 440 kDa AnkB protein is the result of the insertion of a 6.4-kb exon between the spectrin-binding domain (SBD) and death domain (DD). AnkG plays an important role in various excitable tissues. The expression and localization of βIV spectrin and NaV 1.5 were disrupted in mouse cardiomyocytes lacking AnkG, leading to a significant reduction in voltage-gated NaV channel activity. These mice exhibited a decreased heart rate, impaired atrioventricular conduction, increased PR interval, and increased QRS interval (Sato et al. 2011; Makara et al. 2014). ANKB, as well as other ankyrins, consists of four main domains: a membrane-binding domain (MBD), an SBD, a DD, and a C-terminal domain (Figure 3B). The MBD contains 24 consecutive ANK repeats and can interact with membrane proteins such as ion channels and transporters. The SBD contains highly conserved ZU5-ZU5-UPA domains and can interact with βII-spectrin. The function of the DD is still unknown, but reports in the literature suggest that it plays a role in cell apoptosis and inflammation (Park et al. 2007). The DD and C-terminal domain together constitute the regulatory domain, which can directly bind to the MBD and have inhibitory effects (Abdi et al. 2006; Xie et al. 2022).

Although AnkB and AnkG have similar structures, they are associated with different diseases. The specificity of ankyrins is partly determined by the autoinhibitory linker peptide between the MBD and SBD, which prevents AnkB from binding to molecular chaperones (He et al. 2013).

AnkB is widely expressed in both excitatory and inhibitory neurons of the brain, as well as in glial cells (Saunders et al. 2018; Jin et al. 2020). AnkB is considered an axon protein because it is highly concentrated in the axon initial segment (AIS), Ranvier node (NOR), and axon terminals. Within axons, it participates in the transport of organelles, including synaptic vesicles. At the AIS and NOR, AnkB interacts with multiple calcium channels, IP3R, and ion transport proteins (Yoon et al. 2023). Studies investigating the loss of ANK2 function suggest that it is associated with axonal morphology, connectivity, and calcium signaling in excitatory neurons (Yoon et al. 2023; Kline et al. 2014).

Loss of AnkB in mice causes severe neurological defects, such as hypoplasia of the corpus callosum and pyramidal tract and lateral ventricle dilatation (Scotland et al. 1998). Yoon et al. (2023) reported that changes in the synaptic proteome caused by the loss of ANK2 impair neuronal activity and synchrony, leading to behavioral disorders associated with NDDs. Yang et al. (2019) elucidated a mechanism that typically restricts axonal branching: giant AnkB is localized to the periodic axonal plasma membrane domain through its interaction with the L1 cell adhesion molecule protein (L1CAM). This interaction allows AnkB to couple microtubules to the plasma membrane and prevents microtubules from entering nascent axon branches (Chen et al. 2020; Fransen et al. 1997). Furthermore, several studies have suggested that ANK2 mutants exhibit dysregulated intracellular calcium homeostasis and altered expression of calcium channels in excitatory neurons (Yoon et al. 2023; Kline et al. 2014). Homologues of ankyrin proteins are known to interact with ion channels in various types of neurons (Tseng et al. 2015). Loss of AnkB has been shown to lead to decreased expression of voltage-gated calcium channel 2.1 (Cav 2.1) compared to voltage-gated calcium channel 2.2 (Cav 2.2) in the cortex, cerebellum, and brainstem. ANK2 mutations may be associated with seizures (Choi et al. 2019).

We identified a nonsense mutation, c.3007C>T, p.R1003*, in the patient. This mutation results in an amino acid change from Arg to Ter at position 1003 of AnkB, leading to the production of a truncated protein that lacks the subsequent 2955 amino acids due to abnormal translation; this mutation may cause nonsense-mediated decay (NMD) or may prevent interaction with L1CAM, thus preventing the inhibition of axonal growth. Therefore, we propose that this variant is a strong candidate for explaining the patient's clinical phenotype. Additional published data are needed to further support the correlation between the variant and the phenotype (Ji et al. 2019). Other reported variants of the ANK2 gene are shown in Figure 3A.

4 Conclusion

ANK2 gene variants have rarely been reported; to date, only 15 patients have been reported worldwide. Our patient is the 16th patient and the first such patient reported in China. We also summarized the genetic and disease spectra of all 16 patients. ANK2 variants were associated with a series of phenotypes: ASD (9/16 patients); EP (10/16 patients); ASD with EP (4/16 patients); developmental delay and intellectual disability (5/16 patients); poor language communication skills and language and learning disabilities (11/16 patients); abnormal behavior problems (11/16 patients, including anxiety, agitation, mood disorders, attention-deficit/hyperactivity disorder, TD, sleep disorder, self-injury and aggressive behaviour); poor cognition, memory, and adaptability (1 patient); ECG abnormalities and cardiac damage (1 patient); and abnormal EEG results (9 patients). Prompt performance of genetic testing is important for patients with EP in ASDs with intellectual disability, behavioral abnormalities, and EEG abnormalities. ANK2 mutations should also be considered when an exon 27 mutation is detected. There are no specific guidelines or consensus on the treatment of neurological diseases associated with the ANK2 gene. For children experiencing seizures, it is important to develop individualized treatment plans based on factors such as seizure type, EEG, and craniocerebral imaging. For children diagnosed with ASDs, appropriate psychological and behavioral interventions or pharmacological treatments should be implemented according to the corresponding diagnosis and treatment guidelines. For individuals with intellectual disability, attention-deficit/hyperactivity disorder, or TD, it is crucial to conduct prompt evaluations and implement active intervention measures. When necessary, rehabilitation training can be provided to support their needs. In conclusion, ANK2 is a novel gene associated with EP and related neurological disorders, primarily manifesting as ASDs, intellectual development disorders, behavioral abnormalities, and electroencephalogram abnormalities. Genetic testing should be promptly conducted to elucidate the underlying cause, and ANK2 gene mutations should be considered.

Author Contributions

Lu Zhao and Zhi-Dong Qiao wrote the article; Yue-Xin Jia, Jun-Xian Fu, and Tian-Xia Li edited the data; Kai-Ru Jia, Xiao-Fan Yang, and Hao Pan collected the data; Hong Zhao and Jin-Ping Bao improved and modified the article; and Guang-Lu Yang, as the corresponding author, gave comprehensive guidance to the writing of the article.

Ethics Statement

This study was conducted in accordance with the declaration of Helsinki and from the Ethics Committee of the Affiliated Hospital of Inner Mongolia Medical University (No. WZ[2024002]).

Consent

Written informed consent was obtained from the participant.

Conflicts of Interest

The authors declare no conflicts of interest.