New phenotype of severe neonatal episodic laryngospasm due to a missense mutation in SCN4A: A case report and literature review

Abstract

Severe neonatal episodic laryngospasm (SNEL) is an ion channel disease characterized by recurrent life-threatening myotonia of respiratory muscle due to mutations in the voltage-gated sodium channel genes. Here we reported a newborn manifested as paroxysmal cyanosis and limb myotonia after birth. The neonate also developed muscle hypertrophy and stunted growth during the follow-up. Whole exome sequencing confirmed c.2395G>A, p.Ala799Thr heterozygous mutation of SCN4A. Carbamazepine was found to be effective on treating the disease. This case expands our understanding of the phenotype resulting from SCN4Amutations. By summarizing the characteristics of reported 16 cases in SNEL,we found they were mainly in the p.G1306E mutation. The common symptoms were upper airway muscle stiffness and feeding difficulties during neonates.When grow up, most patients have different degrees of recurrent attacks of myotonia and progressed muscle hypertrophy. Some of them have athlete-like special faces but all showed myotonic discharge in eletromyogram.

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Action potentials relies on the function of transmembrane voltage-gated channels that regulate rapid changes in ion fluxes, resulting in muscle contraction. Due to their unique role in the depolarizing phase of action potential, voltage gated sodium channels (VGSCs) are the direct targets of synthetic drugs that modulate cell excitability or the sites of disease-causing mutations^[1]. In humans, the isoform of VGSCs expressed on skeletal muscles is NaV1.4^[2]. Gain-of-function variants in NaV1.4 typically result in myotonia or periodic paralysis^[3]. An infant with severe respiratory muscle myotonic is known as severe neonatal episodic laryngospasm (SNEL).

Skeletal muscle sodium voltage-gated channel alpha subunit 4 gene (SCN4A) mutations are known to cause types of disease, including symptoms of exercise- or cold-induced muscle cramps, stiffness, and myalgias^[4], congenital myopathy ^[5-6], congenital myasthenic syndromes^[7], neonatal stridor or SNEL^[8] . Some of these disorders can be life-threatening.

Here we described a newborn who underwent one neonatal intensive care unit (NICU) and two pediatric intensive care unit (PICU) hospitalizations after birth due to paroxysmal cyanosis and myotonia (including facial muscles, eye muscles, trunk muscles, muscles of the limbs, etc.). At two months of age, he was diagnosed with SNEL by whole exome sequencing, it confirmed that SCN4A heterozygous mutation in c.2395G>A, p.Ala799Thr.

1. Case presentation

A neonate manifested as paroxysmal myotonia with facial cyanosis and clenched hands. He groaned and foamed at the third day after birth and the symptoms lasted for only a few seconds. According to his parent's narratives, he had cyanosis once or twice a day with obvious phlegm in the throat, and cyanosis was aggravated by crying. In addition, episodes with localized swelling around one or both eyes were observed. The swelling would build up over several seconds and then disappear spontaneously (Figure 1). The parents also noticed the paroxysmal unilateral lower extremity jerks when the baby was crying, and the symptom spontaneously terminated within a few seconds. This symptom could happen to each side. At postnatal day 22 after birth, he was diagnosed with neonatal pneumonia and was admitted to NICU for the treatment. After using anti-infection treatment, the sputum volume significantly decreased, but the frequency of apnea was not improved. At postnatal day 33, the patient was diagnosed with epilepsy and congenital cytomegalovirus infection, and was admitted to PICU again where he was treated with oral sodium valproate [40 mg/(kg·d)]. After the treatment, paroxysmal facial cyanosis and myotonia were alleviated. On the 21st day of receiving antiepileptic treatment with valproate, the patient presented a sudden apnea and was followed by a cardiac arrest, possibly triggered by a common cold. Cardiopulmonary resuscitation was performed to save the patient's life. This patient has no apparent family history, and parents also reported no abnormalities during pregnancy or delivery. Physical examination revealed that hypertonia, athlete-like appearance, and mask-like face (Figure 2).

Figure 1. Main symptoms in this case.

A:Right eyelid edema. B: Left eyelid edema. Paroxysmal alternate onset of unilateral eyelid edema lasts for minutes followed by spontaneous recovery. C:Sign of muscular hypertrophy. Generalized muscle hypertrophy and stiffness are mainly located at paraspinal muscles, looked like “a small athlete”.

Figure 2. Patient’s athlete-like appearance before and after treatment.

A and B: Before treatment. The figures of patient shows hypertonia, muscular hypertrophy, athlete-like appearance. C and D: After treatment. Muscle hypertrophy and muscle tension are obviously improved after carbamazepine treatment.

Routine biochemical tests of cardiac, cerebral, pulmonary functions as well as metabolic, blood and urine tests revealed normal findings. Video electroencephalography (VEEG) monitoring revealed several clinical attacks with complexion cyanosis and myotonia. The EEG monitoring demonstrated slightly reduced full conductance voltage during the ictal phase, along with periodic temporal low-medium amplitude slow waves mixed with complex electromyography (EMG) interference. Significant multifocal sharp wave discharges around the temporal region during the interictal phase were present. And EMG results showed muscle rigidity (Figure 3). The patient was subsequently treated by sodium valproate according to a diagnosis of epilepsy. Both cyanosis and myotonia significantly improved after treatment. EEG was performed again and normal results were found during the interictal period, similar as previously.

Figure 3. Different phase of electroencephalogram (EEG) and electromyogram (EMG).

A: Attack phase.EEG revealed slightly reduced full conductance voltage during the ictal phase, along with periodic temporal low-medium amplitude slow waves mixed with complex EMG interference. B: Interictal phase.

Following treatment of low-dose oral carbamazepine [10 mg/(kg·d)], the symptoms include myotonia and apnea were relieved, while sleep quality was significantly improved. Mild paroxysmal facial cyanosis reappeared when the blood concentration of carbamazepine was below 5 μg/mL. When carbamazepine dose increased to 40 mg/(kg·d), muscle tension returned to normal with richer facial expressions,as well as paroxysmal facial cyanosis and myotonia were relieved (Figure 2). In addition, the phenomenon of paroxysmal tremor of unilateral lower limbs when the patient was crying disappeared slowly. However, episodes with localized swelling around one or both eyes still exist.

We did a whole exon gene test for the patient and his parents (Figure 4). It revealed a de novo heterozygous mutation in SCN4A gene, c.2395G>A substitution predicting a p. Ala799Thr missense mutation (Figure 4A), which may lead to protein function variation (Figure 4E-4G). The SCN4A gene test of both parents were normal (Figure 4B and 4C). Although the pathogenicity of this mutation has not been reported previously, pathogenicity and variation of the same position c.2395G>T mutations have been reported in the literature, suggesting its association with SNEL.

Figure 4. Results of SCN4Agene sequence and three-dimensional structure changes of protein.

A: Patient's whole exon sequencing shows c.2395G>A missense mutation in SCN4A gene. B and C: Father and mother's whole exon sequencing shows no variation in this nucleotide site. E and F: Normal protein three-dimensional structure diagram and residue stereogram for alanine at 799 site. G and H: Variation protein three-dimensional structure diagram and residue stereogram for tyrosine at 799 site.

The patient is now 27 months old, and his developmental evaluation at the age of 2 showed that he still could not turn over, climb, or sit alone. His language ability was equivalent to 1 year old level, and the fine movement was at about 15 months old level. He also developed myotonia congenita. The myotonia was most pronounced when movement was initiated but decreased when the movement was repeated several times. It is worth mentioning that the patient had taken carbamazepine at a dose of 40 mg/kg and the dose was reduced to 20 mg/kg 2 months ago. The patient’s athlete-like appearance has been obviously improved (Figure 2).

During follow-up period, the EEG showed that significant multifocal sharp wave discharged around the temporal region at the interictal phase and EMG results showed muscle rigidity.

2. Discussion

We have retrieved 16 cases of SNEL from 2006 to present.All described mutations are missense mutations and affect the gating behavior of ion current passing through NaV1.4. Among them, the p.G1306E mutation accounted for 75%^[8]. Supplementary Table 1 (https://doi.org/10.11817/j.issn.1672-7347.2022.200598T1) has concluded common symptoms in most p.G1306E mutation patients^[9-15]. During neonatal phase, patients are characterized by attacks of muscle stiffness affecting upper airways which causes cyanosis and hypoxemia.They may accompanied with various degrees of feeding difficulties. When grow up, most patients have different degrees of recurrent attacks of muscle stiffness or myotonia and progressed muscle hypertrophy. Some of them have athlete-like faces but all show myotonic discharge in EMG.

The other 4 cases (Supplementary Table 2, https://doi.org/10.11817/j.issn.1672-7347.2022.200598T2) were caused by p.A799S mutation, p.N1297K mutation, p.T1313M mutation, and p.A1156T, respectively^{[8-9, 16-17]}. One case of the p.A799S mutation died of respiratory failure, and one with p.N1297K mutation died of pneumonia at 20 months of age. The developmental milestones of patient with SNEL caused by this mutation delayed slightly, and they could walk independently at 16 to 18 months. Patient with p.T1313M mutation had a slightly delay in the developmental milestones, and was able to walk at 19 months old. Another case caused by p.N1297K mutation is unique. He had difficulties to grasp and hold objects at 18 months, and could sit alone at 18 months old. All the SNEL cases reported have effective response to sodium channel blockers and most of them have good clinical prognosis, except for the death case.

Isolated or recurrent episodes of laryngospasm have a wide range of medical causes, but genetic neurologic or neuromuscular conditions are rarely considered in the differential diagnosis. The features of SNEL are recurrent larynx muscle stiffness and obstructions in the upper airways, and subsequent muscle hypertrophy and myotonia^[18-19]. It's one of severe form of sodium channel myotonia. In this study, we reported a child experienced recurrent abrupt onset of generalized myotonia, following by apnea and cardiac arrest. He had been diagnosed as epilepsy and gastroesophageal reflux, and was not diagnosed as SNEL until 2 months after genetic testing. Both previous studies^{[10, 20]} and the current case were all demonstrated that SNEL was closely related to heterozygous SCN4A gene missense mutations.

SCN4A gene is located on chromosome 17q23-25. It encodes NaV1.4, a subunit of voltage-gated complex which Na⁺ ions pass in accordance with their electrochemical gradient^[21]. In skeletal muscle, normal function of sodium channels is essential for the generation and propagation of action potentials and triggers cellular events that result in muscle contraction. SCN4Amutation is correlated with the activation or inactivation speed of muscle ion channel that changed skeletal muscle excitability^[11]. Dysfunction in skeletal muscle channelopathies may cause a wide range of phenotypes like myotonia congenita, paramyotonia congenita, sodium channel myotonia, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, Andersen Tawil syndrome, and so on.

Myotonia is an abnormal delay in muscle relaxation caused by persistent muscle membrane activity. It can be easily diagnosed by EMG which shows persistent muscle membrane activity during attack phase. Among non-dystrophic myotonias, sodium channel myotonia may be diagnosed in childhood and is related to mutations in the SCN4Agene. To understand the mechanism of laryngospasm and myotonia phenotype of Ala799 in SCN4A mutation, we analyzed the structure and function of its encoded protein. NaV1.4 is made of 4 domains (DI-DIV), and each constituted of 6 transmembrane segments (S1-S6). It has been confirmed that fast inactivation of sodium channel is consistent with II-IV helix of NaV1.4 structure. Through 7 different human sodium channel subunit paralogs, position of alanine 799 in NaV1.4 is topological domain, which is also a part of highly conserved functional motifs. Variation of Ala799 affects the NaV1.4 topology structure, causing NaV1.4 fast inactivation gating channel dysfunction, therefore muscular membrane action potential is impossible to back to rest potential rapidly. Patients appeared muscle cramps, stiffness, myalgias, congenital myopathy or SNEL. Simkin, et al^[22] recorded a typical Na⁺ current traces from TsA 201 cells. The A799S mutant α-subunit channels exhibited a slower decay when compared with WT channels. Overlapping scaled macroscopic Na⁺ current traces clearly showed that persistent current was more prominent in Ala799 mutant than in WT transfected cells. In short, persistant current of Ala799 variants cause abnormal action potential, which accounts for continuous spasm of laryngeal skeletal muscles.

Another characteristic of this disease is secondary muscle hypertrophy. Our patient had a generalized muscle hypertrophy, mainly at paraspinal muscles,and looked like “a small athlete” at 6 months of age. Lion-Francois, et al^[9] also reported that 3 patients with SNEL had muscle hypertrophy at age of 5 month (p.Gly1306Glu), 6 month (p. Gly1306Glu) and at birth (p. Ala799Ser), respectively. Some experts^[19] believe that the cause may be related to muscle acuolar myopathy and T-tubular aggregates. Others^[23-24] consider that muscular hypertrophy may be associated with mutation lied intracellularly between the fourth and fifth transmembrane segments of domain III.

Although the disease is serious, most patients with SNEL caused by SCN4A gene mutation respond well to sodium channel blockers carbamazepine, but don't to diazepam and levodopa. Blockers of the open state of sodium channel can reduce chance of NaV1.4 re-opening and alleviate symptoms. Our patient also showed a significant therapeutic effect after carbamazepine treatment. Unfortunately, the patient 13 in Table 2 died before receiving carbamazepine. It suggests that once infants have symptoms of SNEL and are highly suspected to NaV1.4, they should be treated with carbamazepine in time before genetic results come out.

Neonatal episodic laryngospasm is a serious disease associated with sodium channelopathy. In this study, we demonstrate that new c.2395G>A, p.Ala799Thr NaV1.4 mutation is likely responsible for severe episodic laryngospasm and myotonia. Both the clinical features and laboratory results are consistent with SNEL diagnosis.The variant was pathogenic. Timely identification of this disorder is helpful for effective treatment. The patients with SNEL are suggested preferably be treated with sodium channel blockers as early as possible.

Appendix.

Supplementary Table 1 Related symptoms of p.G1306E mutation in SCN4A gene

Patients	Age of onset and complain	Perinatal disease	Poor sucking/feeding difficult	Respiratory problem	Development delay	Hypo-tonia	Myotonia/Episodic muscle stiffness	Provoked by cold	Muscular hyper-trophy	Other symptoms	EMG revealed myotonia
1[9]	After birth daily stiffness, cyanosis, bradycardia	NA	NA	+ Laryngo- spasm	-	-	+ Handgrip, palpebral, tongue	NA	+ (6 months)	Tonic closure of eyelids (5 months), athletic appearance (6 months)	+
2[9]	3 days, daily episodic apneas	NA	+	+ Obstructive apneas	+	+	+ upper limbs	+	NA	Intermittent stridor (2 month) expression less face, slow limb movements (7 month), athletic appearance	+
3[10]	8 weeks, recurrent ALTEs	Preterm 32 weeks	NA	+ Apena	-	NA	+	+	NA	NA	+
4[10]	6 weeks Episodic cyanosis, choking	Need oxygen but no resuscitation after birth	NA	+ Recurrent laryngo- spasm	-	NA	+ Episodic stiffness Eye closure and hand grip myotonia	+	+ (10 month old) Shoulders and neck	Mild learning difficulties, frequent muscle spasms Eye closure myotonia Short stature with kyphotic posture	+
5[10]	10 days Stridor and feeding difficulties	-	+ Choking with feeds for first 3 years	+ Repeated laryngospasm	-	NA	+ Laryngeal muscle		+ Upper limbs	Herculean appearance and Hulk-like stance	+

Supplementary Table 1 (continued)

Patients	Age of onset and complain	Perinatal disease	Poor sucking/feeding difficult	Respiratory problem	Development delay	Hypo-tonia	Myotonia/Episodic muscle stiffness	Provoked by cold	Muscular hyper-trophy	Other symptoms	EMG revealed myotonia
6[11]	11 years, muscle stiffness	Polyhydr-amnios Fetal hypoki-nesia	+	-	+	+ floppy	+ Lower limb and abdominal muscles	+	+ marked	Arnold-Chiari malformation, hydrocephalus Bilateral hip dysplasia, scoliosis	NA
7-8[12]	after birth, Episodic apneas	Oligohy dramnios, IUGR.	+ GER	+ Episodic apneas	+	+	+ Laryngeal muscle Leg muscle (8 years old)	+	+ Significant	Athletic appearance, Limb movements were limited, diplopia, Strabismus	+
9[13]	15 months, increased creatine kinase levels	-	+ Impaired swallo-wing	+ Respiratory distress, episodic apnea.	-	+ floppy	+ Severe muscle stiffness (6 months)	+	+ General. Mainly paraspinal muscles	Dysmorphic facial, short neck, barrel chest. Forced internal rotation of gleno humeral joint	+
10[14]	First day laryngo spasm, episodic stridor		+ Swallo-wing	+	-	NA	+	+	+ Face and limbs	NA	+
11-12[15]	Early childhood painful muscles	NA	-	+	NA	NA	+ Myotonic stiffness of all muscles	+	-	CK elevated from 957 to 2 170 U/L	+

NA: Not available; CMAP: Compound muscle action potential; EMG: Electromyography; IUGR: Intrauterine growth retardation; GER: Gastroesophageal reflux; ALTEs: Acute life-threatening events.

Supplementary Table 2 Symptoms in other SCN4A gene mutations of SNEL

Patient/Patient No.	Zygosity	Nucleotide changes and Protein changes	Age of onset and complain	Perinata disease	Poor sucking feeding difficult	Respiratory problem	Development delay
this case	Heterozygous	c.2395G>A, p.A799T	3 days, groan and cyonosis	-	-	+ Frequent apena	+
13[9]	Heterozygous	c.2395 G>T p.A799S	After birth, respiratory distress	NA	+	+ Episodic stridor and severe apnea	+
14[8]	Inherited from mother, randfather, great uncle	p.T1313M	24 hours after birth inspiratory stridor	+ Persist stridor	+	+	+ mild
15[16]	De novo heterozygous	c.3891 C> A p.N1297K	Neonatal apnea	NA	+	+ Prolonged apnea. Died at the 20th months (bronchopulmonary infection)	+
16[17]	Inherited from father	c.3466 G> A p.A1156T	35 days, cyanosis and stiffness of lower limbs	-	+	+ apnea	-

Patient/Patient No.	Hypo-tonia	Myotonia/Episodic muscle stiffness	Provoked by cold	Muscular hyper trophy	Other symptoms	Laboratory investigations
this case	-	+	-	-	Swelling eyes, epilepsy, congenital cytomegalovirus infection, athlete-like appearance and mask-like face	EMG results showed muscle rigidity
13[9]	-	+	-	-	Generalized hypertonia, poor reflex movements	EMG revealed myotonic discharge, muscle biopsy showed vacuolar degeneration in fibers
14[8]	-	+ Extra-ocular muscles; Stiffness (2 years)	-	NA	Laryngomalacia, exotropia,	NA
15[16]	+	+ Cold sensitive	+	+	Facial dysmorphism, severe constipation	EMG revealed myotonia.
16[17]	-	+	-	-	Tonic constraction of lower extremities.	EEG captured a focal seizure of left head side

NA: Not available; EMG: Electromyography.

Conflict of Interest

The authors declare that they have no conflicts of interest to disclose.

Note

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