A Novel Missense Mutation in the GLRB Gene Causes Hyperekplexia

Hyperekplexia is a rare disease. Hyperekplexia is characterized by increased muscle tone (hypertonia) and an exaggerated reaction to abrupt unexpected stimuli (Praveen et al., 2001). It affects newborn children and gradually fades during the first year of life. Nonetheless, older patients with hyperekplexia may still startle easily and exhibit stiffiness, which may lead to falls (Russo et al., 2017; Tijssen et al., 1997).

Hyperekplexia results from the dysfunction of the glycinergic neurotransmission system, which is involved in inhibitory neurotransmission. Five genes have been identified as causing the disease (Harvey et al., 2008). Mutations of GLRA1 gene, which encodes the glycine receptor alpha-1 subunit, are the most common (Harvey et al., 2004). However, the glycine transporter-2 (SLC6A5), gephyrin (GPHN), and collybistin, a RhoGEF for the receptor clustering protein, have yet to be identified as contributing to disease (Dafsari et al., 2019; Davies et al., 2010; Harvey et al., 2004).

The detection of these genes is crucial in making a diagnosis for this disease. We here report a case of hyperekplexia resulting from a novel missense GLRB mutation that results in the substitution of methionine at position 99 with lysine (M99K).

The patient was a 31 years old male from Shanghai. He suffered from recurrent falls for 20 years ago, which could be induced by sudden sound and other stimuli. He could not maintain his balance and was easy to fall, resulting in multiple abrasions and sprains. He walked with a wide-based gait for fear of falling. He was conscious at the time of the attacks, and his symptoms disappeared during sleep. He is the only child in his family. His parents were not close relatives, and none of them had similar symptoms. He was born of spontaneous vaginal delivery with a normal birth process and weight (3.2 kg). His intellectual development was normal, but his physical performances were relatively poor during school days.

The physical examination showed the patient had mild hypertension. He presented with a wide-based gait. Tapping his nose tip could induce facial muscle contraction with head back and body stiffness. The tendon reflex of limbs was active. No obvious abnormalities were found in other neurological examinations. The serum creatine kinase level was 412 IU/L (normal range 55-170 IU/L). MRI of the brain with contrast-enhancement revealed an arachnoid cyst in the left temporal area. Ambulatory electroencephalography (AEEG), electromyography (EMG) and other laboratory tests were all normal. Considering the possibility of hyperekplexia, the stiffness and startle symptoms of patients were significantly relieved after treatment with clonazepam.

We collected peripheral blood samples from the proband and his parents. We used the Qiagen kit (QIAGEN, Hilden, Germany) to extract DNA from 3mL blood samples and conduct sample quality inspections. We then used next generation sequencing (NGS) to capture and sequence the exons of 3204 genes related to genetic diseases. The variant was analyzed and classified according to ACMG (the American College of Medical Genetics and Genomics) guidelines (Richards et al., 2015). Moreover, we investigated allele frequency (1000 g, dbSNP, and ExAC) and pathogenicity prediction [Polyphen2 and Mutation Taster (http://www.mutationtaster.org)]. Sanger sequencing was used to confirm the results of second-generation sequencing.

In a humidified incubator with 95% air and 5% CO2, HEK293T cells (ATCC) were maintained in Dulbecco’s modified Eagle medium (DMEM) with 10% FBS and 100 U/mL penicillin/streptomycin. Lipofectamine 3000 (Invitrogen) was used for transfection.

The GLRB wild type and mutant plasmids were obtained from Genechem Company (Shanghai, China). To create site-specific mutants within the CV061 vector, PCR-based site-directed mutagenesis was used. Synthesized and annealed sequences are as follows:

Sense: 5’-ACGGGCCCTCTAGACTCGAGCGCCACCATGAAGTTTTTATTG-3’,

Antisense: 5’-AGTCACTTAAGCTTGGTACCGATAAATATATAGACCAATATATAAC-3’

RNA was extracted from cells with TRIZOL reagent (Invitrogen). PrimeScript 1st Strand cDNA Synthesis Kit (Takara) was used to synthesize the cDNA. SYBR Green Real-time PCR Master Mix (TOYOBO) was used to measure the GLRB level. GLRB mRNA amplification primers were 5′-AGTGCCCCTGGGTATCTTCT-3′ and 5′-CTTTTGGGGTTGTTCAGCAT-3′. GLRB mRNA level was compared to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

GLRB protein expression was determined by Western blotting by using GLRB antibody (1: 1000 dilution, Santa Cruz, sc-365819). GLRB expression was normalized to that of GAPDH (1: 1000 dilution, Cell Signaling Technology, #5174).

After seeding on poly-L-lysine-coated coverslips, HEK293T cells were processed for immunofluorescence. Cells were fixed, eluted with GLRB antibody overnight, and incubated with goat anti mouse fluorescent antibody (P-phycoerythrin, PE) for 30 min. Images were photographed under a fluorescence microscope (Leica).

SWISS-MODEL (https://swissmodel.expasy.org) was an automated server for the modeling of homo- and hetero- protein complexes. UniProt (https://www.uniprot.org) is a free resource for elucidating the amino acid sequence and function of various protein.

One homozygous variant of the GLRB gene, c.T296A (p.M99K), was found in this patient. His parents were both heterozygous carriers of the same variant (Fig. 1). Neither dbSNP (http://www.ncbi.nlm.nih/gov/snp) nor 100 genome project (http://browser.100genomes.org) or Exome Aggregation Consortium (ExAC) contained this variant. By MutationTaster, c.T296A was predicted to cause disease (0.99 probability, range: 0-1.0). With a score of 0.9667, Polyphen-2 also predicted that the mutation is probably damaging (sensitivity: 0.77; specificity: 0.95).

Mutant GLRB’s mRNA level was not affected by real-time PCR (Fig. 2A). There was no difference in WT and mutant GLRB protein expression (Fig. 2B). Furthermore, immunofluorescence indicated that the distribution of mutant protein did not differ significantly from the control (Fig. 2C). Mutation taster showed strong conservation of amino acid sequence near the mutation site between different species (Fig. 3A). We further used SWISS-MODEL software and UniProt to show the mutation site (Fig. 3B).

Hyperekplexia is a relatively rare hereditary nervous system disease, which is related to the dysfunction of the glycinergic neurotransmission system (Zhan et al., 2021). As a result of the lack of understanding of this disease, it is often misdiagnosed as epilepsy or other disorders. So far, a total of 12 patients have been reported in China (Chan et al., 2014; Huang et al., 2018; Li et al., 2017; Poon et al., 2006; Tsai et al., 2004; Yang et al., 2017; Zhan et al., 2020; Zhang et al., 2020). A typical clinical manifestation of hyperekplexia is an excessive startle response after birth. Its inducing factors are often sudden unexpected sound, visual or tactile stimuli, and sometimes even when touching daily operations such as feeding or changing diapers (Koning-Tijssen and Brouwer, 2000). Patients can also have the following abnormal manifestations, such as limb movement during sleep and myoclonus before falling asleep, umbilical hernia or esophageal hiatal hernia, epilepsy, intellectual or motor developmental delay, congenital hip dislocation, and spastic paraplegia (Bakker et al., 2006). Some patients can be complicated with anxiety and depression (Mine et al., 2015). The diagnosis of hyperekplexia is mainly based on clinical manifestations. Detailed history and physical examination are helpful, especially the patient’s neonatal and childhood history. The nose-tapping test can be used as a characteristic diagnostic examination (Shahar et al., 1991). In patients with hyperekplexia, laboratory tests, MRI and EEG are often normal. The diagnosis should exclude the possibility of other diseases. Previous studies suggested that gene mutation detection rates for hyperekplexia were approximately 60% (Davies et al., 2010). Thus, it is necessary to perform genetic analysis for the precise diagnosis. Clonazepam is usually used in treatment, which can compensate for the defects of the GABAergic pathway in the brain stem and enhance the protective response of patients during startle reflex (Zhou et al., 2002). The prognosis of this disease is generally favorable.

At present, at least five genes have been identified to be related to hyperekplexia. GLRA1 is the most frequent mutation gene, encoding glycine transporter subunit α1. GLRB is also a major gene of effect (Chung et al., 2013), which encodes glycine transporter subunit β. GLRA1 binds to GLRB to form postsynaptic hetero pentameric glycine (GlyR) receptor with 2α1:3β subunit (Yang et al., 2012). The presynaptic glycine transporter-2 (SLC6A5) (Alfadhel et al., 2016) as well as two of the GlyR synaptic clustering proteins (GPHN and ARHGEF9) (Rees et al., 2006) have also been identified as having mutations. To date, more than 20 GLRB mutations have been identified as hyperekplexia (Fig. 4) (Chung et al., 2013; Li et al., 2017). Genes encoding GLRB are mutated in many ways, including in-frame deletions, two-hit duplications, large repeats-mediated deletions, and splice site mutations that result in exon skipping and missense mutations (Chung et al., 2013). Inheritance of hyperekplexia can be autosomal dominant or autosomal recessive (Harvey et al., 2008). GLRB mediated hyperekplexia is mainly in the mode of homozygous recessive or a compound heterozygote inheritance (Chung et al., 2013).

GLRB encodes the glycine receptor β subunit which contains an extracellular domain (ECD), a transmembrane domain (TMD) with four α-helices (TM1-TM4), and a cytoplasmic domain as well as glycine receptor α1 subunit. Interestingly, most GLRB mutations are located on the ECD of the protein, not like GLRA1 mutations which were mostly located on the TMDs (Zhan et al., 2020). UniProt showed position 99 of GLRB was also at the extracellular region (https://www.uniprot.org). Recent functional studies indicated some GLRB mutations either reduced the expression of the glycine receptor on the cell surface or reduced the sensitivity to glycine (James et al., 2013). However, our results showed GLRB expression and distribution were not affected obviously. A previous study showed a common mutation M177R that was also located on the ECD did not affect GLRB expression, which was consistent with our results (James et al., 2013). We further used the prediction tool to find the potential pathogenicity of the mutation. Interestingly, the mutation site was highly conserved across species (Fig. 3A), suggesting this mutation may have an important effect. Furthermore, bioinformatics software was performed to illustrate this mutation may be harmful. Since GLRB expression and distribution were unchanged, we hypothesized this mutation may disturb the glycine sensitivity.

In our case, the patient had symptoms of exaggerated startle responses and slightly delayed motor development. Our case suggests that for unexplained abnormal gait and frequent falls in adults, it is also necessary to evaluate the possibility of potential undiagnosed hyperekplexia. We found a homozygous recessive GLRB mutation (c.T296A/p.M99K) in our patient. And this novel mutation did not alter GLRB expression and distribution in HEK293T cells. However, bioinformatics revealed this mutation to be probably deleterious. To date, it is unclear how the mutation detected in our patient altered the function of glycine receptors to cause hyperekplexia. The exact mechanism of GLRB mutation needs to be explored in the future.

We are very grateful to the patient and his parents for cooperating with this study.

The research was supported by the National Natural Science Foundation of China (Grant no. 81801616, 81971535) and Renji Hospital National Natural Science Foundation Youth Cultivation Advancement Project (RJTJ24-QN-060).

This study was approved by the Ethics Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine. Consent was obtained from all participants. The patient and both of his parents were examined in the study.

The authors have declared no conflict of interest.